hello

I have been working with RNA samples for last couple of years. The first and foremost requirement is a good quality RNA sample with least protein and lipid contamination as they tend to reduce the efficiency of PCR. A better alternative is the create cDNA and run you qPCR using the cDNA. Don't forget to elute out the pure cDNA after cDNA preparation is over.

qPCR standard curve is generally generated using a control gene which is supposed to show no variation following your experimental condition. Nevertheless to generate the standard curve, you sample must be pure. For further suggestions on enhancing the sample purity I would like to know the RNA extraction protocol you are following.

You did reverse transcription and the PCR separately, right? If no bands showed up, even for your positive control (using control RNA from the RT kit, I suppose), lots of things could have gone wrong. If you used a control RNA as a positive control, the problem is not RNA quality (even though you MUST have acceptable Abs ratios at 260/280 and 260/230 to do this kind of experiment), and something could have gone wrong with the reverse transcription.

If you have a good amount of RNA, but bad ratios, you should definitely perform a clean-up.

I work with RNA purified from nasal aspirates for viral diagnosis. I always have significant readings at 230 (0.150 to 0.300) (suposedly carbohidrates and solvents) but notorious peaks at 260 (RNA), and my RT-PCRs run well. I think is better to observe an evident peak at 260 that consider readings at 230 if these do not show a peak (rising and decreasing), may be these readings are background such as I think is my case.

like mentioned before, you probably have a contaminant in your RNA that is hindering the PCR reaction such as residual ethanol. if you wanted to, you could probably do a ethanol precipitation, but i would recommend starting from scratch again, and making sure that you remove ALL the ethanol in the washing step.

you need a standard curve to be able to determine the primer efficiency. it tells you how efficient a set of primers work, and is important when you want to compare between primers (eg when you use the 2 delta delta ct method to compare relative quantities of two genes of interest). if the primer for gene A has an amplification efficiency of 99%, while the other primer for gene B has an efficiency of 70%, then it is not reliable to compare between the two primers simply because the products are being produced at different rates. Like Krzysztof mentioned, you dont have to do this in every experiment. you only need to do it for every new primer pair to determine the efficiency. from them on you're okay!

good luck!

Dear Shambhavi,

Sorry for the long post!!! I had the same issue you did when I first started : ) So I hope this is helpful for you.

Before proceeding onto the reverse transcription step, did you Nanodrop or measure the concentration of your RNA? What was your lowest concentration? What kit did you use for reverse transcription? What kit did you use for RNA extraction? What are you extracting? Did you run your samples on agarose gel? Were they degraded? How old are your reagents?

Our lab works with many types of samples (nasal swabs, stools, breast milk, bacterial cells, human cells, and tissues), so when extracting DNA or RNA, I find that different kits extract different samples more efficiently than others. For example, Qiagen RNeasy Plus Mini Kit yields more RNA from stools samples, Qiazol works better on tissues, while the Ambion RiboPure Bacterial Kit extracts RNA better in bacterial cells. This was specific towards my samples, I don't know if these kits would work better for you. However, I do recommend calling the companies to send free kits to extract your samples from. I also find that preheating up the elution buffer before eluting maximizes total RNA yields.

I would also recommend after extracting RNA from your samples to run them on agarose gel. This will give you an idea of whether or not your samples were degraded. RNA are single strand, so they are very sensitive and breakdown quickly. Degraded RNA may affect reverse transcription of RNA to single stranded DNA. The best way to prevent degradation is to keep your samples on ice at all times. Also, handling of samples when extracting RNA is extremely crucial, you also want to spray (prior to extractions) your workspace, bench, and pipets down with 10% sodium hypochlorite, RNAase zap followed by 70% EtOH to remove any RNAses, which can affect the yield of your RNA.

I asked about the concentration about your RNA because usually in reverse transcription kits, they give you a certain concentration amount (e.g 0.5-2 ng) to work with for optimal conditions. I definitely would not proceed on reverse transcription unless you have the minimum concentration recommended by your reverse transcription kit.

Your A260/A230 ratios indicate contamination. This document will give you a better idea. http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf

In regards to your standard curve question, I attached a link (hopefully helpful). http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html