A slew of molecular biology companies have recently introduced kits for extracting and purifying total RNA from cells or tissue for downstream analyses, particularly real-time quantitative PCR, where such a step is crucial to obtaining error-free results.
However, the RNA-purification process can be time-consuming, and researchers are always on the lookout for methods — provided they work well — that can cut down the amount of time spent at the laboratory bench.
Furthermore, according to some, like core lab director Greg Shipley of the University of Texas Health Science Center at Houston, the purification step is not only time-consuming, but may result in some loss or degradation of RNA — an especially salient point when there is little nucleic acid to work with in the first place.
At Cambridge Healthtech Institute's XGen Congress, held March 15-19 in San Diego, Shipley spoke about a method being developed his lab to perform qRT-PCR directly on whole cell lysate preparations.
PCR Insider caught up with Shipley following the conference to discuss the method and how researchers at UTHSCH are using it in their research. Following is an edited transcript of the conversation:
You mentioned in your talk that using cell lysates in RT-qPCR can be both a blessing and a curse. What are some of the advantages and disadvantages?
If one is using sample sizes that are relatively large, whether that's tissue culture, a piece of tissue, or whatever, then it makes sense to make total RNA. However, there are many examples where that is not possible.
My other dream about this whole thing [is that] because everything is in the [cell lysate], you [can] try to take advantage of the genomic DNA that's in there to normalize the transcript data. And in other cases, people just want to look at DNA in general for multiple reasons — for instance, digital PCR.
But I've been having some problems with the methods that we're using right now. I think it's doable — we just haven't hit the right combination of things to do.
Where are the challenges? Is it in sample prep?
Actually, sample prep is incredibly easy. That is one of the blessings of the whole thing. Basically if you're using tissue culture plates, for example, you simply wash the cells with [phosphate-buffered saline] one time, after you get rid of the tissue culture media, and you throw in the lysate buffer, you pipette up and down five times, throw it in the tube, and you're done.
And because you have a very small number of cells, but you may want to do a very large experiment — for instance, you may want to look at multiple PCR targets — you can do that with this technology. That's a big advantage.
From the list I provided during my talk, there are many examples of times when you just don't have very much stuff to work with. Tissue culture is pretty easy pickings. Specific examples [include] stem or primary cells; needle biopsies or fine needle aspirates; bronchial lavage fluid, especially from small animals; sputum; sorted cells from [fluorescence-activated cell sorting] experiments; embryos from developmental studies; [and] laser capture samples from frozen or [formalin-fixed paraffin-embedded] samples.
If sample prep is fairly easy, what have been some of the hurdles thus far?
Well, first off, we've had to figure out whether we're getting exactly what we think we're getting. That's why I did [a] large experiment [using Bar Harbor Biotechnology's] Tox StellARrays — to basically convince myself that everything in [the lysate] was OK, that we didn't have inhibition from carryover protein or whatever that was going to cause a significant problem. [In the experiment, Shipley's lab treated HeLa cells with staurosporine and compared transcripts obtained from purified RNA with those obtained from total cell lysates — Ed.]
And I think I've convinced myself that things look pretty good, at least using the not-quite-released-yet Roche RTR cell lysis system; but I would imagine that if I used something like the Cells-to-Ct kit [from Life Technologies' Applied Biosystems business], it would work equally well.
You mentioned the forthcoming Roche cell lysis kit. Can you discuss this product a little more?
Yes, it's called the Real-Time Ready Cell Lysis kit. They're coming out with a whole line of Real-Time Ready products that includes RT and PCR reagents as well as their own plate arrays. I must disclose that I do work with Roche … so I do get free kits to try.
This kit is in beta testing right now?
Yes, they tell me it will be released in the summer or fall sometime. So far, so good. One of the nice things about it, unlike the Cells-to-Ct kit, is that there is a DNAse step that is critical — especially if your transcript assays don't cross junctions, as not all of mine do; or there are transcripts that don't have junctions, so you don't have any choice there. This DNAse step is folded right into the reverse transcriptase step. I know that seems counterintuitive, but they're using an arctic brine shrimp DNAse, which has about a 5,000-fold larger affinity for double-stranded over single-stranded DNA. Also, they do it at a temperature where I think the reverse transcriptase isn't quite active yet. Their Transcriptor runs at 55˚C, which is actually a pretty toasty reverse transcriptase reaction; but they run the DNase reaction, even though it's suboptimal, at 29˚C to do the DNAse, and then they go on and make the cDNA… boy, the data looks pretty nice so far.
Also, you discussed using the Bar Harbor StellARray product in your staurosporine experiment. Why did you use that product?
I've used the SABiosciences plates before, and they seemed to work well, quite frankly, but Bar Harbor wanted me to try the StellArrays, since they're in direct competition with them, and they're both SYBR Green-based assays. [SABiosciences was acquired by Qiagen in December — Ed.]
They have two things that the SABio product doesn't: One is a matrix that they put the primers in that they believe gives a much better release. I think the SA Bio primers are simply dried to the bottom of the well, although they seem to work. But Bar Harbor claims that they get better, more even distribution of the primers back into your master mix when you put it in the wells. And the second thing is their [Global Pattern Recognition] data analysis, from which I did show data. It allows you to look at the data set without needing a particular gene [or genes] for data normalization. But it does compare the data to 18S rRNA, for example, although that doesn't always work … But for what they're trying to use it for, the vast majority of people that are going to be using it for gene discoveries, sort of a focused microarray using real-time qPCR, their analysis will be perfectly fine. SABio gives you this elaborate Excel spreadsheet for data analysis, but they don't really give you a mechanism to choose the correct normalization genes from their plate, at least the last time I looked. That's a drawback for the naïve user.
Does it make a difference what type of instrumentation you use for qRT-PCR from cell lysates?
No, not really. I showed data from the [Roche LightCycler 480], but I have an [Applied Biosystems 7900 HT Real-Time PCR system], and it would work equally well on there; and it works on my [ABI Prism 7700], as well. However, the excellent temperature uniformity of the block in the LC480 gives the best data for plate arrays and that’s why I use that instrument for these experiments.
What kind of research are you doing, or are others doing, using the cell lysate method?
I am a core lab director, so in theory we don't do research in our lab, although I am doing some. My goal is to bring these technologies into the labs so I can encourage my users to use them. For some of them — for instance, I discussed a FACS experiment with a graduate student [who] was looking at these GFP-labeled lung cells, a subset, and she was going to combine the cells from three mice so she could make total RNA. I said, 'Why do that?' She had 50,000 cells, which is quite a few actually. And I said, 'We could easily make lysates out of these, and then you'd have an n of three instead of an n of one. And we were able to do that. That's just one example.
You want to enable the users, especially clinicians, who are really up on the theory of everything, and the trends and what's going on in the literature, but on the technical side they sometimes don't have a clue, because that's not what they do. It's up to us to enable them to do things they might not have been able to do before.
Also, we currently have an experiment using the Cell-to-Ct [kit] with embryos. We definitely want to give that a try. Those samples are sitting in the freezer now — we just haven't had time to get to them yet. [The investigator's] got 10 embryos in different developmental stages and is treating these mice with a drug that affects implantation. And he wants to know what the mechanism is behind that. He has an idea of some transcripts that might be affected, so we'll look at this at the RNA level first.
Are there situations where it just isn't feasible to use cell lysates in analysis?
I'm sure I'll find situations where there are either tissues or small amounts of something from some source that perhaps have a lot of PCR inhibitors in them. But I must say for the two examples that I mentioned, and from others I've seen — you have to dilute at least 10-fold going into the RT reaction, and then normally you dilute again going into the PCR. If that 10-fold dilution is still going to allow RT inhibitors to mess things up, then that will be a problem.
I'll use hemoglobin as an example. The iron in hemoglobin competes with magnesium, and can mess up a magnesium-requiring enzyme, which is both reverse transcriptase and the Taq polymerase. That's why people don't lyse blood whole and just go for it. There could also be tissues — liver or spleen, for example — that could cause similar problems. And there are myriad other kinds of RT inhibitors out there, and I don't think we even know what they all are, quite frankly. So there might be a chance where you make a lysate like this, and it just has too much of that stuff in there, and it would be a problem. I haven't run into it yet.
Another issue will be fibrous tissues that may not be amenable to optimal disruption by the reagents mentioned above. Another product from a small company, Zygem in New Zealand, has a lysis buffer based on a protease from a thermophile. This product has worked well for us as well but we haven’t tested it extensively yet. It may be a better fit for small fibrous tissue samples.