Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes
Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine...
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Gespeichert in:
| Autor*in: |
Yang, Tao [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2022 |
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| Schlagwörter: |
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| Anmerkung: |
© The Author(s) 2022 |
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| Übergeordnetes Werk: |
Enthalten in: BMC genomics - London : BioMed Central, 2000, 23(2022), 1 vom: 20. Jan. |
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| Übergeordnetes Werk: |
volume:23 ; year:2022 ; number:1 ; day:20 ; month:01 |
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| DOI / URN: |
10.1186/s12864-021-08275-w |
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| Katalog-ID: |
SPR050428802 |
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| 520 | |a Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. | ||
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10.1186/s12864-021-08275-w doi (DE-627)SPR050428802 (SPR)s12864-021-08275-w-e DE-627 ger DE-627 rakwb eng Yang, Tao verfasserin aut Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2022 Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. Detection sensitivity (dpeaa)DE-He213 Formaldehyde-agarose gel (dpeaa)DE-He213 Northern blot analysis (dpeaa)DE-He213 Posthybridization wash (dpeaa)DE-He213 Zhang, Mingdi aut Zhang, Nianhui (orcid)0000-0002-3663-6495 aut Enthalten in BMC genomics London : BioMed Central, 2000 23(2022), 1 vom: 20. Jan. (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:23 year:2022 number:1 day:20 month:01 https://dx.doi.org/10.1186/s12864-021-08275-w kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 1 20 01 |
| spelling |
10.1186/s12864-021-08275-w doi (DE-627)SPR050428802 (SPR)s12864-021-08275-w-e DE-627 ger DE-627 rakwb eng Yang, Tao verfasserin aut Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2022 Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. Detection sensitivity (dpeaa)DE-He213 Formaldehyde-agarose gel (dpeaa)DE-He213 Northern blot analysis (dpeaa)DE-He213 Posthybridization wash (dpeaa)DE-He213 Zhang, Mingdi aut Zhang, Nianhui (orcid)0000-0002-3663-6495 aut Enthalten in BMC genomics London : BioMed Central, 2000 23(2022), 1 vom: 20. Jan. (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:23 year:2022 number:1 day:20 month:01 https://dx.doi.org/10.1186/s12864-021-08275-w kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 1 20 01 |
| allfields_unstemmed |
10.1186/s12864-021-08275-w doi (DE-627)SPR050428802 (SPR)s12864-021-08275-w-e DE-627 ger DE-627 rakwb eng Yang, Tao verfasserin aut Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2022 Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. Detection sensitivity (dpeaa)DE-He213 Formaldehyde-agarose gel (dpeaa)DE-He213 Northern blot analysis (dpeaa)DE-He213 Posthybridization wash (dpeaa)DE-He213 Zhang, Mingdi aut Zhang, Nianhui (orcid)0000-0002-3663-6495 aut Enthalten in BMC genomics London : BioMed Central, 2000 23(2022), 1 vom: 20. Jan. (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:23 year:2022 number:1 day:20 month:01 https://dx.doi.org/10.1186/s12864-021-08275-w kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 1 20 01 |
| allfieldsGer |
10.1186/s12864-021-08275-w doi (DE-627)SPR050428802 (SPR)s12864-021-08275-w-e DE-627 ger DE-627 rakwb eng Yang, Tao verfasserin aut Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2022 Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. Detection sensitivity (dpeaa)DE-He213 Formaldehyde-agarose gel (dpeaa)DE-He213 Northern blot analysis (dpeaa)DE-He213 Posthybridization wash (dpeaa)DE-He213 Zhang, Mingdi aut Zhang, Nianhui (orcid)0000-0002-3663-6495 aut Enthalten in BMC genomics London : BioMed Central, 2000 23(2022), 1 vom: 20. Jan. (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:23 year:2022 number:1 day:20 month:01 https://dx.doi.org/10.1186/s12864-021-08275-w kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 1 20 01 |
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10.1186/s12864-021-08275-w doi (DE-627)SPR050428802 (SPR)s12864-021-08275-w-e DE-627 ger DE-627 rakwb eng Yang, Tao verfasserin aut Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2022 Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. Detection sensitivity (dpeaa)DE-He213 Formaldehyde-agarose gel (dpeaa)DE-He213 Northern blot analysis (dpeaa)DE-He213 Posthybridization wash (dpeaa)DE-He213 Zhang, Mingdi aut Zhang, Nianhui (orcid)0000-0002-3663-6495 aut Enthalten in BMC genomics London : BioMed Central, 2000 23(2022), 1 vom: 20. Jan. (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:23 year:2022 number:1 day:20 month:01 https://dx.doi.org/10.1186/s12864-021-08275-w kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 23 2022 1 20 01 |
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modified northern blot protocol for easy detection of mrnas in total rna using radiolabeled probes |
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Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes |
| abstract |
Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. © The Author(s) 2022 |
| abstractGer |
Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. © The Author(s) 2022 |
| abstract_unstemmed |
Background Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use. Results In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment. Conclusions The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA. © The Author(s) 2022 |
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| title_short |
Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes |
| url |
https://dx.doi.org/10.1186/s12864-021-08275-w |
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| author2 |
Zhang, Mingdi Zhang, Nianhui |
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Zhang, Mingdi Zhang, Nianhui |
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| doi_str |
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2024-07-03T15:28:53.552Z |
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