Dear Lisa, 

Basically 1 micro gram of RNA is used to make cDNA (50 microliter rxn).I have also used 500 nanogram for making cDNA and it worked well. Based on my experience I think you need more RNA.

Hi Lisa,

I'm afraid, as Nitesh and Laurence comment, that you need at least 500 ng RNA (1ug is commonly used) to perform a good cDNA synthesis. You must know that more than 90% of your material correspond to rRNA leaving a small fraction of sample to represent the expression of the whole genome of your model organism. So, if you want to detect a reliable expression from coding genes you must increase the RNA input fro cDNA synthesis.

Good luck!

In my lab we routinely use 500 ng total RNA for a 20 µl reaction. However, we also tried 250 ng and 100 ng, which both also worked fine. Thus, if the amount of total RNA is limited for some reason, not so much total RNA is really required.

I just wonder why the concentration of your RNA is so low. Frequently, this is sign of problems with RNA purification and might also be an indication of RNA with a low quality. Did you carefully check the quality of your RNA?

RNA purity and RNA integrity are most important. If there is any problem with the RNA purification, contaminants such as genomic DNA (gDNA), DNases, RNases and proteases can end up in the final sample and lead to total or at least partial degradation of RNA and/or DNA, (partial) degradation/denaturation of enzymes, or amplification of gDNA.

Carrying out your experiments with substandard RNA can have dramatic effects on your results.

There are several methods for evaluating your RNA before proceeding to the RT step. If you assess the quality by absorbance, the RNA preparation should have a minimum A260/280 ratio of about 2. However, make sure that you don’t work at the detection limit of your spectrophotometer because in that case the results are not reliable.

If you have access to an Agilent Bioanalyzer system (Agilent 2100) this might be the best choice. The Agilent Bioanalyzer uses microfluidics to size‐separate and quantitate RNA in a highly reproducible way (electrophoresis and flow cytometry).

it depends on the the level of expression of genes that you plan to study.

usually within range 100-1000 ng for reverse transcription you get reliable results for majority of genes.

However, if amount RNA is limiting factor and you do not want to go through RNA amplification step before PCR you still can go as low as 10-20 ng ang get meaningful results for a lot of genes

Armin,

The cells that were treated with compounds were not very happy so therefore there were simply a much lower number of cells to harvest RNA from. The trizol was also 1 ml for a  6 well plate and frozen at -80C.

It depends on your cDNA synthesis kit. I just made qPCR with cDNA generated from 50 ng total RNA. It worked just fine for my genes of interest. Just keep in mind qPCR will likely not be reliable for low-expression genes in this case.