What should be the concentration of RNA used for cDNA synthesis considering the analysis of low expressing genes in the template.
Should the concentration be maximum 1ug or can exceed beyond that?
Most protocols suggest to use between 0.1 to 1 ug RNA. Up to 5 ug can be used but I am not sure about it. With experience we have learnt that good quality LiCl -precipitated RNA makes the difference and sometimes adding too much RNA is counter productive.
Dear Mansi: We have used 0.2 pg of RNA for cDNA synthesis and total RNA amount of starting material can vary from 0.1 pg to 5 μg of total RNA
Thermo scientif SuperScript™ III First-Strand Synthesis System SuperMix is an optimized SuperMix formulation for first-strand cDNA synthesis from purified poly(A)+
https://www.thermofisher.com/sa/en/home/references/protocols/nucleic-acid-amplification-and-expression-profiling/cdna-protocol/first-strand-cdna-synthesis-from-total-rna-or-poly-a-rna.html
For RNA to cDNA synthesis, we use 1 ug of RNA, then the final (20 ul) reaction mix is added to 80 ul of diH2O (final cDNA concentration 1:5), and our data is beautfiful for qPCR / rt-PCR.
You can use 2 ug of total RNA for cDNA synthesis which is good enough for further qPCR studies.
Hi! I'm having the same doubt. If I want to use 5 ug or more of RNA for DNA synthesis, do I still use 200 U (as instructed by manufacturer on data sheet, for 2 ug) or should I scale it up? I'm using the Promega M-MLV RT. Thank you!!
Amount of RNA The amount of mRNA in an assay can be as high as 2 μg; the amount of total RNA up to 20 μg. We recommend using at least 1 μg of total RNA in a 40 μl reaction volume. Enzyme concentration The ratio of Reverse Transcriptase AMV (50 U/assay) to RNA is not critical up to 20 μg total RNA.
You can go upto 2 ug of RNA for cDNA synthsis and use 1 uL for qRT-PCR to quantify low expressing genes. (For most of the genes 1:5 dilution of cDNA works well)
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts