We tried to observe the quality of human RNA from tissues by agarose gel and incubation of RNA at 70C for 10 min prior to gel loading. However, we see only one spot or smear.
RT-PCR of bactin was OK.
Thanks for your hints
Hi there:
1. Try denaturing your RNA by heating as you have done but add 10% formamide to more fully eliminate secondary structure: This will culminate in sharper bands in the absence of RNA degradation. You could even try running the old style formaldehyde agarose gels typically utilised for Northern blotting but that is probably too much work for simple visualisation of integrity. 10% formamide in your loading buffer however will improve sharpness for analysis on a standard agarose gel
2. I agree with Petra that standard spectrophotometry will tend to be dominated by unincorporated nucleotides if present. Nevertheless 260/230 ratios in particular will tell you something about the potential presence (or not) of impurities: In particular, a 260/230 ratio of less than 1.0 implies potentially high and adverse levels of either salts - and specifically and usually the GITC present in initial lysis buffers like RLT and the first was buffer in commercial RNA extraction kits or alternatively if extraction is performed with phenol or Trizol bleed through of organics into your final RNA eluate.
3. RLT is present in these buffers to provide a stable environment for RNA following initial lysis by inhibiting RNAses and in the case of the first wash buffer to denature protein resident on a purification column before washing with a second ethanol based buffer to desalt your prep before final elution. If however GITC bleeds through into the final eluted RNA it can also inhibit down stream enzymes like Taq and RT (in addition to its prescribed job of inhibiting RNAses)
4. To minimise salt contamination and in particular GITC after adding ethanol based washed buffers to a column wait 1 minute before spinning through and then repeat. I do this routinely and find this helps with keeping the 260/230 ratio > 1.0 (and preferably >1.5)
5. In the case of phenolic/trizol extraction if this ratio is < 1.0 simply ethanol precipitate your RNA; & wash with 70% ethanol. This tends to bring back the ratio above 1.0. When precipitating incidentally, apart from adding 1/10 sodium acetate pH 4-5 and 3 x volumes of ethanol if you add 1ul of anything from 1mg/ml to 20mg/ml molecular grade glycogen you will end with a larger more opaque pellet and then makes it easier to manipulate and in particular allows you to wash with 70% ethanol by vortexing which will facilitate desalting and thus removal of GITC
6. Alternatively, for best quality RNA especially with larger tissue masses it is common practice to perform initial trizol chloroform extraction and then take the upper aqueous phase; mix with an equal volume of 70% ethanol and then subject to a column based extraction method. Generally speaking this will tend to give the best RNA quality of all
7. If you have already performed a column based extraction methos and discover your 260/230 ratio is < 1.0, simply mix your eluate with an equal volume of 70% ethanol and column purify again. I have always found this brings the 260/230 ratio from < 1.0 to > 1.0 and sometimes renders RNA samples that were not fit for purpose, e.g. failed to amplify or make cDNA efficiently into samples that then did
8. I will finish by saying that providing your 260/280 ratio is > 1.6-1.7 then with a 260/230 ratio of > 1.0 and in the absence of significant degradation RNA tends to work well. A lot of people tend to get fixated with the 260/280 ratio whereas for downstream utility the 260/230 ratio is often more important
the use of high concentration of guanidium could be increase RNA yield.
Ghamartaj,
It would be helpful to add a ladder to your gel so we could get an idea about the size of the fragments, but it does not look good.
I'm not quite clear what you are saying and asking... I assume you mean BAD quality RNA despite good peak and ratios on a spectrophotometer?
There are two options here:
a) You actually have good quality RNA, but your gel and buffer contained lots of RNases, so your RNA degraded while it was running on the gel.
b) Spectrophotometer readings DO NOT say anything about RNA quality. They simply detect the concentration of nucleotides in the solution, but DO NOT mean that these nucleotides are actually part of DNA or RNA sequences. So in this case, you never actually had good quality RNA (likely if tissues are not flashfrozen immediately or stored in RNAlater).
Hi there:
1. Try denaturing your RNA by heating as you have done but add 10% formamide to more fully eliminate secondary structure: This will culminate in sharper bands in the absence of RNA degradation. You could even try running the old style formaldehyde agarose gels typically utilised for Northern blotting but that is probably too much work for simple visualisation of integrity. 10% formamide in your loading buffer however will improve sharpness for analysis on a standard agarose gel
2. I agree with Petra that standard spectrophotometry will tend to be dominated by unincorporated nucleotides if present. Nevertheless 260/230 ratios in particular will tell you something about the potential presence (or not) of impurities: In particular, a 260/230 ratio of less than 1.0 implies potentially high and adverse levels of either salts - and specifically and usually the GITC present in initial lysis buffers like RLT and the first was buffer in commercial RNA extraction kits or alternatively if extraction is performed with phenol or Trizol bleed through of organics into your final RNA eluate.
3. RLT is present in these buffers to provide a stable environment for RNA following initial lysis by inhibiting RNAses and in the case of the first wash buffer to denature protein resident on a purification column before washing with a second ethanol based buffer to desalt your prep before final elution. If however GITC bleeds through into the final eluted RNA it can also inhibit down stream enzymes like Taq and RT (in addition to its prescribed job of inhibiting RNAses)
4. To minimise salt contamination and in particular GITC after adding ethanol based washed buffers to a column wait 1 minute before spinning through and then repeat. I do this routinely and find this helps with keeping the 260/230 ratio > 1.0 (and preferably >1.5)
5. In the case of phenolic/trizol extraction if this ratio is < 1.0 simply ethanol precipitate your RNA; & wash with 70% ethanol. This tends to bring back the ratio above 1.0. When precipitating incidentally, apart from adding 1/10 sodium acetate pH 4-5 and 3 x volumes of ethanol if you add 1ul of anything from 1mg/ml to 20mg/ml molecular grade glycogen you will end with a larger more opaque pellet and then makes it easier to manipulate and in particular allows you to wash with 70% ethanol by vortexing which will facilitate desalting and thus removal of GITC
6. Alternatively, for best quality RNA especially with larger tissue masses it is common practice to perform initial trizol chloroform extraction and then take the upper aqueous phase; mix with an equal volume of 70% ethanol and then subject to a column based extraction method. Generally speaking this will tend to give the best RNA quality of all
7. If you have already performed a column based extraction methos and discover your 260/230 ratio is < 1.0, simply mix your eluate with an equal volume of 70% ethanol and column purify again. I have always found this brings the 260/230 ratio from < 1.0 to > 1.0 and sometimes renders RNA samples that were not fit for purpose, e.g. failed to amplify or make cDNA efficiently into samples that then did
8. I will finish by saying that providing your 260/280 ratio is > 1.6-1.7 then with a 260/230 ratio of > 1.0 and in the absence of significant degradation RNA tends to work well. A lot of people tend to get fixated with the 260/280 ratio whereas for downstream utility the 260/230 ratio is often more important
Laurence Stuart Hall said:
"Try denaturing your RNA by heating as you have done but add 10% formamide to more fully eliminate secondary structure: This will culminate in sharper bands in the absence of RNA degradation. You could even try running the old style formaldehyde agarose gels typically utilised for Northern blotting but that is probably too much work for simple visualisation of integrity. 10% formamide in your loading buffer however will improve sharpness for analysis on a standard agarose gel"
I agree with what Laurence said above, but don't try to cut corners and do it in the easiest way possible at this point, especially if you have problems . I would recommend full Formaldehyde-MOPS gel system. Here is a sample protocol:
http://oomyceteworld.net/protocols/RNA%20blot%20(formaldehyde).pdf
The relevant Sections are A-C, blotting is not necessary for your purposes. At a minimum, prepare your sample as described in the protocol and run on a regular 1XTBE or TAE agarose gel.
Nice comment Anton about the MOPS running buffer
The full Northern gel will give you fully Denatured linear RNA molecules which will therefore run exclusively according to size:
I will also add or remind protagonists that agarose is actually a colloid NOT a true solid with a defined structure; unlike say acrylamide bis gels. Consequently, resolution is only down to about 20bp even with linear species and if you add to that secondary structure caused by incomplete denaturation this will inevitably cause smeraring that has nothing to do with bona fide degradation
By denaturing the sample with heat + 10% formamide and subsequently running samples through a denaturing (formaldehyde gel) secondary structure will be minimised or eliminated allowing users to better discern RNA degradation
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