Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
<p>Abstract</p> <p>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-...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2007-10-01
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| Series: | Plant Methods |
| Online Access: | http://www.plantmethods.com/content/3/1/12 |
| _version_ | 1811259862332473344 |
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| author | Walton Eric F Wood Marion Wu Rongmei Varkonyi-Gasic Erika Hellens Roger P |
| author_facet | Walton Eric F Wood Marion Wu Rongmei Varkonyi-Gasic Erika Hellens Roger P |
| author_sort | Walton Eric F |
| collection | DOAJ |
| description | <p>Abstract</p> <p>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.</p> |
| first_indexed | 2024-04-12T18:37:58Z |
| format | Article |
| id | doaj.art-70c0b06fb8134673b5136d7d929a7fe1 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-04-12T18:37:58Z |
| publishDate | 2007-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-70c0b06fb8134673b5136d7d929a7fe12022-12-22T03:20:52ZengBMCPlant Methods1746-48112007-10-01311210.1186/1746-4811-3-12Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAsWalton Eric FWood MarionWu RongmeiVarkonyi-Gasic ErikaHellens Roger P<p>Abstract</p> <p>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.</p>http://www.plantmethods.com/content/3/1/12 |
| spellingShingle | Walton Eric F Wood Marion Wu Rongmei Varkonyi-Gasic Erika Hellens Roger P Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs Plant Methods |
| title | Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs |
| title_full | Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs |
| title_fullStr | Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs |
| title_full_unstemmed | Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs |
| title_short | Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs |
| title_sort | protocol a highly sensitive rt pcr method for detection and quantification of micrornas |
| url | http://www.plantmethods.com/content/3/1/12 |
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