RNA extraction and preparation - RTqPCR - (Sep/01/2011 )
I'm new to posting on these forums and also a new PhD student this year, so forgive me if I do not explain my questions clearly straight off! :-) I have a couple of queries about sample preparation for RTqPCR, which I have limited previous practical experience with, aside from the theory.
Firstly, I use the QIAGEN RNeasy mini kit to extract the RNA from my mammalian cells and have previously managed to get good A260:280 ratios and yield. However, although I have used the same protocol (with the optional steps of DNase digestion and dry spin included, as suggested from the useful advice on these forums), the RNA I have obtained this time round are generally all at a really low yeild (mainly below 2.5 ng/ul, but a few at my usual ~200 ng/ul plus).
I have been wondering if the low yeild is to do with the higher elution volume, but can anyone suggest what I can do to improve the yeild in future experiments so I can get enough of the sample for cDNA synthesis and RTqPCR in duplicate? Also, although the A260:280 's are not too bad for this round of RNA extracts at 1.7, the A260:230 's are very low. Does this mean my samples are contaminated and I need to go back to the cell cultures to do the RNA extractions again?
Any advice you can give to me as a lowly PhD newbie would be very much appreciated!
p.s. can anyone also recommend some good resources for the practical side of RTqPCR and for primer efficiency correcting Ct values? Thank you.
Hi Science Badger, welcome to BioForum!
So you isolated RNA from cultured cells and only got 2.5 ng/ul RNA. How many cells did you use or from what sized vessels were the cells? Did you scrap off the cells after adding RTL buffer? What is the elution volume? The concentration of 2.5 ng/ul is really low unless you also used a very low number of cells. This can be improved by increasing the number of cells and decrease the elution volume, for example, from 50 to 30 ul. The low A260:230 value may indicate contamination with salts or phenol or protein in the RNA solution. Apart from protein contamination, pH value in the water used to dissolve RNA also affect A260:280 ratio. You can read more on this page http://biomedicalgenomics.org/RNA_quality_control.html
For general qPCR technical issues and primer efficiency you can read Suzanne Kennedy's blogs at http://bitesizebio.com/articles/10-tips-for-consistent-real-time-pcr-rtpcr/
Thanks for the warm welcome and for your response!
I cultured my cells in a T-25 and they were approximately 60-70%; so I would say there were around 2 x 106 cells. The procedure I used was identical to that provided by Qiagen with the optional dry spin and DNase 1 treatement steps inlcuded. After removing the media, I added RLT buffer (350 ul) then used a cell scraper before pipetting the lysate into the QIAshredder column and then proceeding with the RNeasy protocol. The final elution volume was 50 ul, which I did twice as from previous RNA yeild from with these particular cells and stored the extracts at -80C. Do you think I should stick with a 50 ul elution in future?
With regard to the A260:230 ratios, it looks like the samples I do have are salt/phenol contaminated and therefore unusuable. Do you have any suggestions to reduce this kind of contamination with future preparations? I already ensure I RNase zap the work bench, use nitrile gloves and autoclave pipette tips just used for RNA, but any recommended further precautions I could take would be greatly appreciated!
Thanks for sending those useful links, I shall look through them this afternoon :-)
If you have such low RNA concentration, the 260:230 ratio will be IMHO always bad, there are residual salts or whatever that are constant, but if you have extremely low concentration and thus the 260, that skewes the ratio.