I'm currently having some issues obtaining blood RNA with a high enough 260:230 value for RNAseq. My protocol is as follows;
1. Blood RNA was initially extracted via the Ribopure Blood extraction kit
2. This was then passed through the GlobinClear RNA kit to remove the bulk of globin transcripts.
3. The resultant Globin depleted RNA was passed through the RNeasy clean up protocol from Qiagen.
I've been measuring the quality of the RNA between each step, with my initial 260:230 values from the Ribopure kit averaging around 2.00. This dips after the Globinclear kit to ~ 1.3. However, when an attempt is made to purify using the RNeasy cleanup column based protocol, this value dips down to sub 1.00.
I'm relatively confident the source of 230nm absorbance is the Guanidine salt in the RLT lysis buffer. I've tried extending the length of time I wash with RPE, the number of wash steps, and also ensuring I coat the entire column with the RPE so as not to leave out any residual salts but nothing appears to be improving my 260:230 ratios. My thoughts are whether or not I could simply leave out the RLT lysis buffer step as I'm not too sure of its purpose when used on already isolated RNA?
Any suggestions would be much appreciated!
EDIT: I should have mentioned, we're hoping to mimic the same protocol as one previously ran by my supervisor, who had no problems with 260:230 values after RNeasy cleanup. Therefore it would be great to try and find a way to use the kits listed before looking elsewhere for cleanup techniques.
If possible do a PAX gene (BD/Qiagen) based RNA extraction, hope you will not get such kind of problems.
Dear Alex,
I used to have the same problems with the RNeasy micro kit. I also tried all kinds of optimization (extra washing steps, filling up the column to make sure everything is washed...) but I never got good A260/A230 ratios.
I think the RLT buffer is needed for the RNA to bind to the column. I tried to purify the RNA without the RLT buffer and it didn't work. I finally changed to the RNA clean and concentrator kit -5 (Zymo research), which delivered highly pure RNA.
Good luck!
Dear Alex,
I second Theodora's suggestion to use Zymo kits. I was using RNeasy for a long time and those ratios get a little finicky at times, although there were usually good. I usually do a LiCl cleanup when the bad ratios happened but the integrity of the RNA is sometimes lost. When I tried Zymo the first time, I felt like I should have used Zymo from the start!
Thank you for your speedy responses guys! I think I agree it may well be worth pursuing other methods of cleaning up these samples.
Hello Alex! my work-partner experienced this issue before.
That buffer made issues and generate poor ratios of 260/230. I have some tips that can improve the purity of RNA.
RLT lysis buffer should be at 30-35C, verify it.
Before put your elution buffer to the column (final step), with a pipette and tip try to remove the salt residuos that are stuck inside of column, just around the border of membrane,
Use the elution buffer (i prefer molecular grade water) at 70C.
If you still getting poor ratios (i do not think so) you can purify your RNA with sodium acetate method
http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids
you gonna make it ;)
Rafa
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