Dear Sulail

• your 260/280 ratios are good and your recovery at 1ug/ul is also excellent. Hence you strong peak at 260nM
• your 260/230 ratios of less than 1.0 indicate salt contamination
• since your yields are excellent and your 260/280 ratios are acceptable - indicating low protein contamination - your main issue is how to eradicate the extra salt; indicates by the 260/230 ratio < 1.0
• i think in part you are on the right track that the problem is partly related to the amount of starting material: howver a recovery of 1ug/ul is not an excessive concentration; indeed it is ideal
• Your white pellets though do suggest salt especially with your 260/230 ratio 1.0 and ideally >1.5 and therefore that your just obtain a strong peak at 260nm without that initial shoulder corresponding to strong absorbable at 230nm
5 votes 3 thanks

You are getting chemical contamination from extraction chemicals, that is what the 260/230 ratio indicates. DEPC is for inhibiting RNases so adding more of that won't help.

The 260/280 is for proteins, and they are okay-ish.

Some methods such as microarrays are sensitive to 260/230 as the carryover chemicals from the extraction can increase background, but for PCR i don't recall having issues with lower 260/230 ratios. I worked with ischemia-reperfused tissue and i highly doubt those ratios result from that.

https://tools.thermofisher.com/content/sfs/brochures/TN52646-E-0215M-NucleicAcid.pdf

260/230 indicate contamination.

260/280 represent protein contamination.

Best,

Umair

The results show carry over contamination and dont bother you go ahead with cDNA synthesis, you will get results for qPCR, take care when you use cDNA product for PCR dilute it to 1: 3 or 1: 5 with nuclease free water for its not possible quantify cDNA and secondly for uniformity use equal concentratons of RNA for cDNA synthesis.. like in your case keep it 500 ng/ul

Dear Sulail, A260/230 ratio is used to check the purity of extracted sample.

For RNA, an ideal A260/230 is above 1.5 in general. Chemical / contaminants from extraction procedure always contribute significantly for 230 nm range, if the extraction was not done properly and carefully. Therefore, it results in low A260/230 values, as indicated in your data. Such common chemicals are EDTA, Phenol, Thiocyanate, GITC, BCP, chloroform, salts and Glycogen. You can look into your extraction protocol to check which chemical(s) are involved in your case and you need to tackle it down, as they may interfere with reverse transcription phase and your cDNA won't be able to help you much in qPCR round then. Hence, A260/230 values are much important for downstream applications and should not be ignored.

Don't forget to make sure that you blanked your nanodrop properly with the same buffer or elution solution, you have used finally to elute your RNA, as it is a quite common mistake seen more often.

But, it is important to know that apart from contamination case, there are few more factors contributing to this low A260/230 ratio, which everyone must keep in their minds like;

• Less amount of extracted RNA in samples.
• Fragmentation of RNA.
• Possible DNAse contamination.
• Calibrating nanodrop, while its pedestal is dirty or not cleaned since long.

So as a rule, if your RNA sample A260/280 comes less than 1.4 then re-precipitate RNA or revise your extraction and if the A260/280 is near 1.7 for the RNA, so use lesser RNA to make cDNA both volume wise and concentration wise.

Regards.....

Dear Sulail

• your 260/280 ratios are good and your recovery at 1ug/ul is also excellent. Hence you strong peak at 260nM
• your 260/230 ratios of less than 1.0 indicate salt contamination
• since your yields are excellent and your 260/280 ratios are acceptable - indicating low protein contamination - your main issue is how to eradicate the extra salt; indicates by the 260/230 ratio < 1.0
• i think in part you are on the right track that the problem is partly related to the amount of starting material: howver a recovery of 1ug/ul is not an excessive concentration; indeed it is ideal
• Your white pellets though do suggest salt especially with your 260/230 ratio 1.0 and ideally >1.5 and therefore that your just obtain a strong peak at 260nm without that initial shoulder corresponding to strong absorbable at 230nm
5 votes 0 thanks

Thank you all for putting up your suggestions. Dr. Hall, you are always a lifesaver. I need to know about the temperature for spinning. Like for steps 1, 3, 5 and 8. Am I supposed to centri at 4 degree Celsius like the instruction manual says? Or RT

4C always better for RNA but if you don't have a refrigerated centrifuge room temp also ok

Perfect! Thanks once again

Dear Stuart

For an understanding, the samples 5,7,8,9 there is shift in peak towards 270 and an extra peak in 230. Is these samples are phenol contaminated? and other samples 4 and 6 have clear peak at 260 (but extra peak at 230). What is the possible contamination causes this kind of figures?.

We most often seen this in our sample, if we isolated RNA from cells seeded on scaffolds.

Thank you for helping us. All your suggestions are really helping us lot.

Hello everyone.. i also need your suggestions over qRT_PCR question. please follow the link.

https://www.researchgate.net/topics

Dear Stuart and other scholars,

I followed the protocol & the results for RNA using nanodrop are as follows:

Can you please add a comment.

Sample---Conc.--- 260/280 --- 260/230

1-------------563.7--------1.98-------------2.27

2-------------583.1--------1.99-------------1.89

3-------------808.8--------2.03-------------2.22

4-------------163.2--------2.03------------1.95

5------------514------------1.95-----------------2.1

Sample 4 conc. is relatively low? can this be a problem? Certainly I will normalize all of the samples before making cdna.

The values for the ratios are within normal limit or have exceeded?

Many thanks!!

They are fine, don't worry about sample 4 because typically 500ng-1ug is sufficient for most mRNA transcripts, which means at most you may pipette 5-10uL. Again, just because some value is not within the range does not mean it is unsuitable for RT-qPCR. A nice thing that would make your life easier would be to dilute a part of your RNA samples to 100ng/uL which would make multiple cDNA conversions much easier as you would always pipette the same for each sample.

Agreed

Since I will be using DNase, I will have to accomodate 1ug in 10ul as the protocol says. After that I will be picking same amount for each.

Thank you. Without you guys, I don’t know what I would have done.

Dear Sulail

• That is, after chloroform/Trzol extraction, subjecting the pellet to 1-2 washes with ROOM TEMP 70% ethanol and vortexing (as decsribed in my previous answer for Sulail)
• Alternatively, if you are purifying RNA with columns, when you add the first column was buffer containing ethanol (which is there to wash away such salt before finally eluting your purified RNA) add the buffer, wait 1 min (to allow the ethanol to percolate fully into the silica matrix of the column; spin; then REPEAT this wash: By doing this your more effectively deslat your RNA prepe residentr n the column (viz. completely remove GITC) and this willbe refeected in 260/230 ratios > 1.0 (and ideally > 1.5 as now obtained by Sulail)

Thank you 😊. Desalting was magical. Happy to hear all is well now.