Problems tracking down problems...RAR!

[brainsnomnom]

I am currently beating my head against the wall, but I'm going to stop long enough to type. So, I am attempting to do qPCR to look at gene expression for 4 genes of interest in cell cultures under various environmental conditions. Previous work with another cell line (same species, same cancer, just different line) showed good amplification of all 4 genes with nice single PCR bands of the expected size. Cue nightmare: changed cell lines, now even when extracted RNA looks good on the Nanodrop, with the exact same run conditions/primer concentrations/etc., I am getting amplification of housekeeping genes at much higher Cts than control spleen cDNA, and amplification of nonspecific crap for genes of interest. Frustrated trying to figure out whether it's inhibition of the RT reaction by carryover products from the RNA extraction that are not showing up on the Nanodrop, degradation of RNA (I have an Amresco rapid formaldehyde-free gel kit on the way, won't be here for another week and a half sadly), or inhibition of qPCR from RT carryover. Using the 5 Prime Perfectpure RNA extraction kit with on-column DNAse step, BioRad iScript cDNA synthesis kit, and SYBR green on a BioRad CFX 96 cycler. I am so frustrated I want to scream, and can't understand why changing cell lines would make me have to reinvent the wheel! Argh. Here's a short list of methods I have tried, to no avail:

Lysis: have tried collecting/spinning/flash-freezing in liquid nitrogen/-80, then lysis before thawing, have tried scraping in the can and collecting, have tried lysis into the can directly followed by scraping

RNA extraction: Tri-reagent, Tri-reagent with ethanol precipitation/wash (lost almost all of my sample), 5 prime kit (column-based, no chaotropic salts unless you count lithium chloride)

cDNA synthesis: Quanta q-script, Bio-rad iScript: both are oligo-DT/random hexamer primed, MMLV, rnase H+

I'm a clinician-researcher, so time is forever at a ridiculous premium, and money is approaching zero at this point. We don't have a Bioanalyzer. Is the most sane thing to do just to look at RNA on the gel and if it's not degraded assume some carry-over is inhibiting RT? Then maybe another ethanol precipitation? This crap is making me want to take a long walk off a short pier.

[mchlbrmn]

I suggest running a dilution series, in duplicate or triplicate, to determine the actual primer efficiency. You can also look carefully at the curve and if the efficiency is too high in the most concentrated samples, but OK in the dilutions, this suggests an inhibitor. If the efficiency is good, this may suggest your PCR works but you lack good template. If the efficiency is good when concentrated, then too high in dilutions, this suggests non specific secondary bands coming up later. If this happens much later than your experimental Cts, this wouldn't really affect the data, and would suggest that the nonspecific crap bands come up at after excessive cycles after you experiment is effectively already complete, and they don't compete eith experimental bands. If the efficiency is too low, this suggests a problem with PCR conditions (well, you are trying 4 genes, so that's not so likely in all 4?).
For example, if you have substantial genomic DNA contamination, the Nanodrop OD would be good, but PCR Cp would be late for amplicons inhibited by intron that only work well on cDNA. (DNAse removal methods are known to sometimes be imperfect.) The more complex genomic DNA template might cause more secondary bands, as you are seeing. Did you run a no-Reverse Transcriptase control? If a NRT control is positive, this indicates genomic DNA (or other) contamination.
Were your housekeeping controls single melt peaks? It is possible, but I guess not likely for 4 genes, that by switching tissues your gene of interest could have alternate splices in the new tissue that could cause additional band/s.
Well, those are my thoughts.

[relaxin]

I think your problem is low RNA yield. You can check on the quality of RNA by running a regular TBE agarose gel. If you see two prominent rRNA bands, then the RNA would be fine. Include equal amount of RNA from the first cell line for comparison.

I noticed some cell lines exppress different levels of genes, even the housekeeping ones. So the conditions you worked out for the first cell line may not work for the second one.

Do you use random primers for RT? They will work even if the RNA is partially degraded.

[brainsnomnom]

@mchlbrmn: do you mean cDNA dilution curve or RNA dilutions going into the RT reaction? My yields on the spec are around 700-900ng/ul, so it's pretty diluted in a 20ul RT reaction. I did a mini-dilution curve (1:8, 1:4, 1:2, 1, 2x template) with my cDNA and got some dilution-responsive inhibition in my HKG, and for my GOI when I ran it out on a gel I got incorrectly sized bands and when sequenced it was gobbledegook. Not getting anything real in NRT and NTC wells. I include a normal spleen cDNA as control in each run just to prove that my primers are (still) working, and they are, so I think it is a template problem. This last run cells were pelleted and stored in RNAlater, but previously I have lysed directly in the can with similar results. Primers are designed around an intron, but I do an on-column DNAse step anyway. Is it worth enriching for poly-A prior to RT step? I'm scared I'm going to lose everything. Did I mention these cells are a pain in the a$$ to grow. Wish we had a Bioanalyzer...

@relaxin: both cDNA kits used are a combination of oligoDT and random hexamers; You can run a regular TBE gel for RNA? Do you have a protocol for this? I have ordered some of Amresco's formaldehyde-free super-fast gels but they must be making them in magical Oompah-loompahland because they are going to take 2 weeks to get here. Meanwhile, the clock ticks. Wonder if it's worth going back to the first batch of RNA and doing an ethanol precipitation using pellet paint so I can see the imaginary pellet. Oy.

[mchlbrmn]

I was talking about determing the PCR efficiency, so that would be dilutions of the PCR template (cDNA (or plasmid)).

"700-900ng/ul" No problem.

If you run a cDNA, and a 1/10 dilution, and there is a 3.3 cycle increment, this indicates that the PCR is working efficiently. (Unless the first sample is delayed by contaminant inhibition, and the second is delayed by poor PCR efficiency from those primers or the PCR conditions, and the two delays cancel each other out to giv a normal 3.3 cycle increment. That's why it's a bit better to run a more full dilution series so you see the result after contaminants are diluted out.) I normally run all experimental samples diluted 1/10. What efficiency does your software calculate for the dilutions you did? Did the dilution match the result?

"I did a mini-dilution curve (1:8, 1:4, 1:2, 1, 2x template) with my cDNA and got some dilution-responsive inhibition in my HKG" Are you saying even your positive shows inhibition? Oh, so you know the PCR conditions are not good? If it's from contaminant inhibition, then the efficiency should improve on dilute samples, but maybe you need to be more dilute than 1/8. As I said, I normally run all my samples diluted 1/10. I have seen inhibition if I don't dilute. (Also, it pushes the experimental Cp's back so they will be more likely to be in the standard curve range.)

"Not getting anything real in NRT and NTC wells." If you are getting the same bands as your experimental samples in the NRT, but not the NTC, this could indicate genomic contamination. If you have genomic, then your PCR may be failing for lack of RNA template (DNA would be quantitated by OD instead of the RNA). If you get bands in the NTC negative, and it shows up with a short enough Cp to interfere with your experimental PCR results, this indicates that your primers/PCR conditions are not good (?). There should be no contamination in the NTC, right? This would be unrelated to template.

What are the Cp's of the negatives, the spleen, and the experimentals? You have been running undiluted cDNA? How much RNA concentration in RT? How much RT rxn in PCR?

Is it possisble that the cell just doesn't express the genes. (Or expresses a splice your primer doesn't see?) Did you run a positive primer set for your troublesome experimental samples? A gene that should definitley be expressed in sufficient quantity and is PCRed with known good primers? (Or, is that what you thought you were doing?)

I assume the intron is large enough not to be PCRed, and that isn't one of the bands you saw.

I did polyA selection long ago and haven't checked recent protocols, but you probably need a lot of RNA to purify mRNA. Whole RNA is usually good enough to use.

I gotta go now.

[relaxin]

[quote="brainsnomnom]
@relaxin: both cDNA kits used are a combination of oligoDT and random hexamers; You can run a regular TBE gel for RNA? Do you have a protocol for this? I have ordered some of Amresco's formaldehyde-free super-fast gels but they must be making them in magical Oompah-loompahland because they are going to take 2 weeks to get here. Meanwhile, the clock ticks. Wonder if it's worth going back to the first batch of RNA and doing an ethanol precipitation using pellet paint so I can see the imaginary pellet. Oy.[/quote]

Yes, I did this once when I was a postdoc, because my mentor told me to do. It is just a regular agarose DNA gel. It is a "quick and dirty" way to confirm the quality of RNA. You may try it with some other RNA first.

If you use the co-precipitant, you may not be able to determine the concentration of the RNA by Absorbance again.

[brainsnomnom]

The co-precipitant comes with a correction factor, so you have to measure at 260 and at 600 on the nanodrop and then do a little mathematical correction. So they say... but I would love to hear if anyone has experience with this.

So, do you have a picture of what that kind of gel should look like? I'm guessing it has the same 18s/28s bands and some other stuff? Do you use DNA loading dye or something RNA specific? Would running it at higher voltage on a 4% gel get tighter bands?

[relaxin]

I do not remember all the details. But I think it was run just like a regular DNA gel. The ethidium bromide stained gel looked like a smear with two strong bands of rRNA.

[procopio f.a.]

let me try:
so you performed qpcr for HKG and 4 genes of interested with a cell line and all work fine.
the same assay on another cell line give you super bad results, high ct for HKG and almost nothing for your specific genes.
right?

first at all i would like to check if it s some stochastic or systematic.
perform rna extraction, cdna synthesis and pcr for the 2 cell lines in parallel. This just to confirm that your problem is new cell line related and not some broken in your kits.

If you get good results for the old cell line but not for the new one then is some related to the rna that you get in the second cell line. How much is the difference in ct for the HKG between the 2 cell lines???? let say you have 10 ct difference, then you should use 2^10 of the new cell line to get the same amount of signal.
Ofc 2^10 is a huge number, i m just suggesting to use more cell for the new cell line actually.

For rna extraction i normally use filter columns (rna aquous ambion), not really a great fun of trizol, EtoH precipitation etcetc whatever you use try to re suspend your rna in a super low volume, in order to check if you get better results.

pfa

[mchlbrmn]

If you run RNA on a normal DNA type gel, make sure you have no RNAse contamination. My lab used to add RNAse to running dye for running out mini plasmid preps (to remove tRNA before the kits), so if we ran RNA in those gel boxes without cleaning them up first, the results weren't good. It's probably a good idea to clean out the gel box thoroughly first. (What with? I'm not sure. A specific RNAse remover if you have it, or mayb a paste of SDS, then lots of rinsing? You can search the web.)

[brainsnomnom]

This forum is completely awesome, and I really, really appreciate all the great advice. I am beginning to wonder if my problem might not be lipid inhibition of my RT reaction? I have been reading that the column kits, unless specifically designed for lipid tissues are not good for this, and it can interfere with reactions without showing up on Nanodrop measurements. I didn't really think it was going to be a problem since I'm using established cultured glioblastoma cell lines, which I figured can't possibly be as bad as dissociated brain tissue with regard to lipid content but maybe there is enough there to cause problems? The last time things worked well I had done a Tri-reagent extraction (well, actually, the super grad student in the lab did it for me while I watched), so maybe these guys are just needing some phenol-based lovin' to get rid of lipid. Will try that and see what happens. Has anyone had good luck with the gel phase separation tubes that make a gel layer between aqueous and organic layers? My hands shake a lot and I seem to be super bad at that step, getting a lot of carryover lowering my 260/230 (which is why I switched to columns in the first place!). Thanks again for the wisdom and input.