Dear User
1. I am currently part of a research project where total RNA is extracted from tiny fractions of patient clinical samples. Consequently, we are getting 50ng to 100ng of total RNA:
2. We want to make cDNA with superscript III and then perform real time qPCR with sybr green for specific genes of interst
3. However, I have never really used much less than 200ng of total RNA to make cDNA for sybr green qPCR so wanted to gauge from other users what my real options are; Let me tress these are patient samples so obtaining more stsrating material to boost the amount of RNA for RT PCR to > 200ng is not an option. Accordingly,
4. Can I relaible make cDNA for quantitative purposes using 50ng of total RNA: This is in 30ul of eluate so we would have to reprecipitate to have this amount in 8ul for the purposes of setting up a 20ul RT reaction
5. If not do we need to pre amplify our RNA before conversion to cDNA ?
6. I was wondering if we could take our existing kit for miRNA and just add a linker adaptor (and not the Poly A tail required for mIR as we are intersted in mRNA with endogenous Poly A tails) or are kits for mIR pre amplication by poil a linkers plus 5' adaptors optimsed for pre amplification of 20bp mIR rather than 1-3kb mRNA species ?
This is alot to consider but I would appreciate any help that can be provided to one or more parts of this question
Kind Regards
https://www.researchgate.net/post/Does_anyone_have_experience_on_cDNA_preamplification_systems_for_RT_qPCR_application
"Lowest amount of total RNA that can be reliably used to make cDNA for sybr green qPCR ?"
There is no precise answer for this as it depends on many variables, PCR can detect as little as a single molecule.
I also wouldn't recommend pre-amp but permissible in some situations (have a read of page 104 - http://www.gene-quantification.com/reiter-pfaffl-qpcr-optimization-2011.pdf ). You should also stick to manufacturer's loading instructions since they have data about its optimal performance, and yes the amount of RNA input can influence the result. Generally speaking, more is better, but "most" is not necessarily the best.
With Superscript III pg of RNA can be reliably used for cDNA conversion, however if the amount will be useful for detection through primers would depend on the abundance of the transcript in your sample too.
Instead of precipitating the RNA to redissolve in smaller volume, simply increase the total volume of the reaction so that you can use the whole 30ul in the reaction. This can help to avoid loss of precious samples.
Personnally I will think that preamplification would not really be necessary and may lead to artefacts due to the amplification process itself.
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