I am a beginner at this. I have recently begun isolating RNAs from differentiated T cells and my efficiency has been the worst! I really don't intend to waste any reagents or sacrifice many more animals just to improve my efficiency!
Anyway, prior to starting the experiment, I had treated MilliQ water with DEPC for 2 hrs ( as suggested by the recipie of Cold Spring Harbor) and autoclaved it twice before storing it at room temperature. Then, the differentiated cells were harvested and pelleted to resuspend in 500uL of Trizol by repeatedly pipetting the mixture up and down. Thereafter, I let it stay for 5 min at room temperature before adding 0.1mL of chloroform. I inverted the contents of the tube for 15s-20s. Then after 2-3 min, I spun it at 12000 rpm for 15 min at 4°C. Then, I transferred the the aqueous layer carefully into a new tube. And then added o.25mL of Isopropanol to it. I allowed it to stand for 15 min at room temperature again before centrifuging at 12000 rpm for 10 min. I DIDN'T get any pellet (perhaps because of the low cell count). So I continued with the protocol wherein I added 0.5mL of 75% ethanol to the supposed pellet and then vortexed plus inverted it for few seconds. Then, I spun it at 12000 rpm for 5 min at 4°C. After washing, I placed the tubes inverted to let all of the alcohol away. After 25 min or so, I added the DEPC treated double autoclaved water.
From the research scholar who guided me with this protocol 2 months ago, o have been told that I hadn't inverted the tubes vigorously. That could be possible. But can that cause a great deal of problem with the purity as well ??? I have been getting the absorbance values of 260/260nm in absolutely absurd and unimaginable range - 0.15 to 0.39 or something !! And the yield is obviously low - somewhere around 1.36 - 1.56.
I meant 260/230nm reading for purity and 260/280nm for the yield.
For RNA extraction, it should be a very clean environment in terms of nucleases. You better use some RNAase away solution to clean pipettes and banch before do anything. You need to work fast and try to use the optimized protocol for specific cells to not waste time and animals etc.. It is little bit tricky. I would read more from researchgate before I keep going. I would suggest you to read qiagen RNA mini kit protocol. There are many helpful info in it. I hope it helps. Best luck...
Dear Akriti Baby Kanth,
Your problem is a typical when small amount of starting material is used for RNA extraction.
A low 260/230 ratio is indicative of guanidine salts that are used in TRIzol carry over into the sample or organic inhibitors (such as humic acids or polysaccharides if the sample is environmental). For low 260/230 readings, the best approach is to try more washes of the RNA sample. Moreover, low overall A260 values give probably unreliable A230 values. So, if you have a small amount of cells and low RNA yield all your values and ratios become shaky and every little bit of contamination becomes "overrepresented".
A low 260/280 reading from protein carry over most likely occurred because of using too much sample for the kit or method. The sample overwhelmed the chemistry and the proteins were not completely removed. Try cleaning up the sample with another round of your method- either phenol:chloroform and precipitate or add the binding salts and ethanol. Next time, try using less sample and make sure homogenization is complete.
In general, I can suggest you to precipitate your RNA samples (accorging to the protocol attached). I have used this approuch in my work and it works great (for improving both 260/280 and 260/230 ratios). You can also try to follow the recomendations given by Stefan Krebs et al (attached article).
At the same time, according to QIAGEN Newsletter (March 15, 2010, page 7) discussing the effect of low 260/230 ratios in RNA preparations on downstream applications it was found, that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications (https://www.qiagen.com/us/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en).
If you have any questions, please ask it!
Best regards,
Maxim
The RNA extraction protocol has already been standardized in the lab. That's why I fear about my efficiency. But thank you so much sirs for your suggestions ! I shall definitely look into them and follow them while extracting RNA from low cell count! Thank you !
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue