Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used...
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Autor*in:	Varkonyi-Gasic, Erika [verfasserIn] Wu, Rongmei Wood, Marion Walton, Eric F Hellens, Roger P

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2007

Schlagwörter:	Reverse Transcription Primer Pulse Reverse Transcription Minus Reverse Transcription Control Sanger Institute miRBase

Anmerkung:	© Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007

Übergeordnetes Werk:	Enthalten in: Plant methods - London : BioMed Central, 2005, 3(2007), 1 vom: 12. Okt.
Übergeordnetes Werk:	volume:3 ; year:2007 ; number:1 ; day:12 ; month:10

Links:	Volltext

DOI / URN:	10.1186/1746-4811-3-12

Katalog-ID:	SPR029403960

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520			\|a Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.
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allfields	10.1186/1746-4811-3-12 doi (DE-627)SPR029403960 (SPR)1746-4811-3-12-e DE-627 ger DE-627 rakwb eng Varkonyi-Gasic, Erika verfasserin aut Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007 Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213 Wu, Rongmei aut Wood, Marion aut Walton, Eric F aut Hellens, Roger P aut Enthalten in Plant methods London : BioMed Central, 2005 3(2007), 1 vom: 12. Okt. (DE-627)500321191 (DE-600)2203723-8 1746-4811 nnns volume:3 year:2007 number:1 day:12 month:10 https://dx.doi.org/10.1186/1746-4811-3-12 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA 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_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 3 2007 1 12 10
spelling	10.1186/1746-4811-3-12 doi (DE-627)SPR029403960 (SPR)1746-4811-3-12-e DE-627 ger DE-627 rakwb eng Varkonyi-Gasic, Erika verfasserin aut Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007 Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213 Wu, Rongmei aut Wood, Marion aut Walton, Eric F aut Hellens, Roger P aut Enthalten in Plant methods London : BioMed Central, 2005 3(2007), 1 vom: 12. Okt. (DE-627)500321191 (DE-600)2203723-8 1746-4811 nnns volume:3 year:2007 number:1 day:12 month:10 https://dx.doi.org/10.1186/1746-4811-3-12 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA 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_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 3 2007 1 12 10
allfields_unstemmed	10.1186/1746-4811-3-12 doi (DE-627)SPR029403960 (SPR)1746-4811-3-12-e DE-627 ger DE-627 rakwb eng Varkonyi-Gasic, Erika verfasserin aut Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007 Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213 Wu, Rongmei aut Wood, Marion aut Walton, Eric F aut Hellens, Roger P aut Enthalten in Plant methods London : BioMed Central, 2005 3(2007), 1 vom: 12. Okt. (DE-627)500321191 (DE-600)2203723-8 1746-4811 nnns volume:3 year:2007 number:1 day:12 month:10 https://dx.doi.org/10.1186/1746-4811-3-12 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA 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_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 3 2007 1 12 10
allfieldsGer	10.1186/1746-4811-3-12 doi (DE-627)SPR029403960 (SPR)1746-4811-3-12-e DE-627 ger DE-627 rakwb eng Varkonyi-Gasic, Erika verfasserin aut Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007 Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213 Wu, Rongmei aut Wood, Marion aut Walton, Eric F aut Hellens, Roger P aut Enthalten in Plant methods London : BioMed Central, 2005 3(2007), 1 vom: 12. Okt. (DE-627)500321191 (DE-600)2203723-8 1746-4811 nnns volume:3 year:2007 number:1 day:12 month:10 https://dx.doi.org/10.1186/1746-4811-3-12 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA 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_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 3 2007 1 12 10
allfieldsSound	10.1186/1746-4811-3-12 doi (DE-627)SPR029403960 (SPR)1746-4811-3-12-e DE-627 ger DE-627 rakwb eng Varkonyi-Gasic, Erika verfasserin aut Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs 2007 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007 Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213 Wu, Rongmei aut Wood, Marion aut Walton, Eric F aut Hellens, Roger P aut Enthalten in Plant methods London : BioMed Central, 2005 3(2007), 1 vom: 12. Okt. (DE-627)500321191 (DE-600)2203723-8 1746-4811 nnns volume:3 year:2007 number:1 day:12 month:10 https://dx.doi.org/10.1186/1746-4811-3-12 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA 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_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 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 3 2007 1 12 10
language	English
source	Enthalten in Plant methods 3(2007), 1 vom: 12. Okt. volume:3 year:2007 number:1 day:12 month:10
sourceStr	Enthalten in Plant methods 3(2007), 1 vom: 12. Okt. volume:3 year:2007 number:1 day:12 month:10
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Reverse Transcription Primer Pulse Reverse Transcription Minus Reverse Transcription Control Sanger Institute miRBase
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container_title	Plant methods
authorswithroles_txt_mv	Varkonyi-Gasic, Erika @@aut@@ Wu, Rongmei @@aut@@ Wood, Marion @@aut@@ Walton, Eric F @@aut@@ Hellens, Roger P @@aut@@
publishDateDaySort_date	2007-10-12T00:00:00Z
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author	Varkonyi-Gasic, Erika
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topic_title	Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs Reverse Transcription Primer (dpeaa)DE-He213 Pulse Reverse Transcription (dpeaa)DE-He213 Minus Reverse Transcription Control (dpeaa)DE-He213 Sanger Institute miRBase (dpeaa)DE-He213
topic	misc Reverse Transcription Primer misc Pulse Reverse Transcription misc Minus Reverse Transcription Control misc Sanger Institute miRBase
topic_unstemmed	misc Reverse Transcription Primer misc Pulse Reverse Transcription misc Minus Reverse Transcription Control misc Sanger Institute miRBase
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title	Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
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title_full	Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
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title_sort	protocol: a highly sensitive rt-pcr method for detection and quantification of micrornas
title_auth	Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
abstract	Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007
abstractGer	Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007
abstract_unstemmed	Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression. © Varkonyi-Gasic et al; licensee BioMed Central Ltd. 2007
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title_short	Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
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