RNA extraction troubleshooting
Read through the manufacturer’s guide for the extraction before beginning.
RNase free environment: Before beginning any experiment that involves the use of RNA, ensure the table, gloves and equipment are RNase free.
This can be achieved either by using an RNase spray solution, or working in clean hood that has been sterilized with alcohol and UV for 20 mins.
Ensure the tips and pipettes to be used are DNase and RNase free, dedicated for RNA use.
You may use DEPC treated tips and tubes or tips that have filters.
Optimal Cell lysis: A primary reason for low yield of RNA, is the cells were not lysed optimally.
This can occur due to high cell density for lysis. Consult the manual to understand the maximum limit for the kit.
The cell pellet was not loosened prior to adding the lysis buffer. If the pellet isn’t loosened, the buffer cannot efficiently lyse the cells as the cells will clump and the nucleic acid material will be shrouded by lipids and proteins which will not be picked up by the column, leading to loss of RNA.
(Recommended) Perform the cell lysis on ice immediately or store the sample with lysis buffer in -20 degree celsius or -80 degree celsius for 2 days to a week before continuing with the isolation.
This works as cooler temperatures will reduce the activity of any RNase present in the sample.
Use a higher volume of the cell lysis buffer, as cell density isn’t always the accurate measure of the efficiency of lysis.
Low A260/280 ratio: This indicates protein contamination in your sample.
Refer to the kit for guidance, as each kit has its own method for this step. However, a general idea is to increase the incubation of the Proteinase K with the sample.
Low A260/230 ratio: This indicates residual guanidine salts.
This can be avoided by following the wash steps as mentioned.
Allow the column to stand with the wash buffer for 2 to 3 mins to ensure optimal results.
Ensure the liquid in the flow-through does not come in contact with the column, this will also case a loss of RNA.
DNA contamination: Genomic DNA contamination could occur when there is too much sample or the DNase treatment was not effective.
Use less sample or check with its guide to check the higher limit of the sample concentration for the column.
Perform the DNase treatment twice, once in the tube before adding the sample to the column and again in the column.
Poor RNA yield:
Ensure RNA is not degraded by following steps mentioned in RNase free environment and optimal cell density.
Allow the elution buffer to stand in the column with sample to have optimal elution of RNA(minimum 2 mins).
The RNA performance is low: Due to high salt and ethanol in the eluted RNA
Ensure that the column does not come in contact with the follow-through. In case this occurs, centrifuge the column. (Recommended: before elution, perform an extra centrifugation step to ensure the column is dry prior to elution.)
As a general note, do not use pipettes for mixing (except when adding samples to the columns), mix the sample and reagents using gentle vortex. This reduces sample loss from residual volumes in the tips.
Also ensure all reagents are mixed well before use, are all at the required temperature and are not contaminated.