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cDNA was synthesized from 100 ng total RNA, ..
The following protocol is designed for large tissue samples (tissue volume 10-100 μl), which normally yield about 10-100 μg of total RNA. Smaller amounts of starting material (expected to yield about 1 μg RNA or less) should be prepared in the same way, with the exclusion of the second phenol-chloroform extraction (step 4) and LiCl precipitation (step 6). Additionally, the final "pellet" should be dissolved in 5 μl instead of 40 μl of water and transferred directly to cDNA synthesis, omitting the agarose gel analysis.
We suggest the following procedure for total RNA isolation based on years of experience working with complicated samples and small amounts of starting material (Matz, 2002). The procedure is suitable for all types of tissues from wide variety of animal species. The method is based on the well-known protocol of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987), except that all steps are performed at neutral pH instead of acidic as was originally suggested. Also, we precipitate the RNA with lithium chloride (LiCl) for increased stability of the RNA preparation and improvement of cDNA synthesis.
Total RNA isolation protocol - PrimerDigital
A Highly linear cDNA synthesis reactions. Total RNA was purified from HeLa S3 cells using the miRNeasy Mini Kit. cDNA was prepared in a miScript II reverse transcription reaction using HiSpec Buffer for a range of RNA amounts from 10 pg to 2 ï¿½g. Each cDNA was then used as a template in real-time PCR reactions using a miScript Primer Assay for 3 mature miRNAs (miR-16, miR-20a, and miR-21) and the miScript SYBR Green PCR Kit. This data demonstrates the broad dynamic detection range that is achievable when using the miScript II PCR System. B Synthetic miR-21 was used to generate cDNA using the miScript II Reverse Transcription Kit under both the HiSpec and HiFlex buffering conditions. A range of amounts from 10 copies to 106 copies of this cDNA was used in real-time PCR using the miScript PCR System. Real-time PCR was performed on the Rotor-Gene Q real-time PCR insturment. The resultant CT values decreased linearly with increasing miRNA copy number, indicating sensitive detection from a wide range of template amounts.
Molecular Biology Protocols: Total RNA isolation protocol ..
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