I am trying to do an RNA extraction of H9C2 cell cultures - these are rat cardiomyocytes. So far, we have found very low yield of RNA each time (around 1-2ng), despite following the instructions meticulously. We went through the troubleshooting tips that come with the kit, and so far have increased the concentration of lysis buffer, incubated the spin columns with RNAse free water before the final centrifugation step, and increased sonication time. However, we have only managed to up the RNA yield to around 7ng. Can anyone suggest anything we may have missed? I am entirely new to PCR so it may be something obvious that I've not realised.
How many cells do you use to make lysate? Increase the number of cells and maybe try trizol extraction following manufacturer's protocol (Invitrogen). goodluck
If you are using a cell line, you probably don't need to use the RNeasy micro kit. It is easy to overload the micro columns and block them with crud from the lysate, thereby preventing RNA binding to the filters in the micro columns. I would suggest using the RNeasy lipid kit which has a trizol step built in (called Qiazol by Qiagen, but effectively the same thing).
Have you tried passing the lysate through a Qia Shredder column to reduce viscosity before extracting RNA?
I agree with previous comments and I can add one more thing. How much water did you use for elution at the end? Try to use minimum volume of water for elution.
Which method did you use for RNA concentration measuring? I recommend you nanodrop or Fluorometer.
Thanks for the answers everyone!
The first things we tried were increasing cell number, increasing the amount of lysis buffer and allowing a ten minute incubation with RNAse free water for the final elution, none of which made a difference. Each time we have been using nanodrop for measuring RNA concentration.
What finally did it however was using an iCell protocol for cardiomyocytes - it uses the same Qiagen kit, but with a few added steps such as using the Qiashredder column mentioned by Amanda, and adding in 100% ethanol washes and a proteinase K treatment. After using this we managed to get a good yield of 100-250 ng with a really nice purity.
Thanks to all the helpful comments!!
- Badges
- Science method