I have encapsulated my stem cells inside alginate hydrogel, I usualy use Thermofisher algimatrix dissolving buffer (https://www.thermofisher.com/order/catalog/product/A1134001) for isolating my cells from hydrogel and extracting RNA using Qiagen Rneasy mini kit but the problem is that each time I get very low A260/230 ratios below 1 and also vey low RNA yied about 2-3 ng/ul. The reason for lower RNA yield might be due to lower encapsulated cell number(~ 200000) but I can't find the reason for low A260/230 ratio, is that due to using hydrogel? how can I clean up my samples to increase the ratio?Also can I proceed to qPCR with this low A260/230 ratio?
Dear Sevda
I think You need to improve the yield. Having 2-3 ng/uL (I assume RNA dissolved in max 10 uL volume) is very low, and perhaps represents poor or even failed extraction. Since RNA concentration calculated using OD at 260, having more RNA will likely improve 260/230 ratio by itself. I can be wrong, but I would first try to work on yield. If You will still have low 260/230 ratio after improved yield (at least 10-times) I would think about clean up procedure.
230 nm is related to GITC-Phenol contamination. To eliminate these compounds, wash the pellet with 75% ethanol twice and then after the centrifuge remove all of the remained ethanol and evaporate the last bit of it at room temperature.
If you are using column kit, repeat your last wash step twice as this reagent contained ethanol as well.
Depends on how low it is!! Also you’re doing cDNA then you will dilute it a lot. So hopefully it should be fine :)
Hi Sedva,
I'm currently troubleshooting a similar RNA yield issue as well. I found that when I'm running the RNeasy Plus (or normal RNeasy) kit that repeating the washing steps with RPE buffer greatly improved the A260/A230 ratio by removing all the compounds used to free the RNA and ends up co-purifying on the membrane. Maybe give that a whirl with some standard RNA that you know you will get a good yield with. 2-3 ng/µL is exceptionally low so keep in mind that if you have a contaminant issue its't going to be relatively very pronounced in a yield that small than if your yield was in the µg or total RNA.
Good Luck and post back with your findings.
Mike
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