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FDA Researchers Describe Inexpensive Reagent Enabling RT-qPCR of Crude Cell Lysate

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NEW YORK (GenomeWeb) — Scientists at the US Food and Drug Administration's Office of Vaccines Research and Review have developed a simple, inexpensive, cell lysis buffer for use in cell cultures to generate crude lysate appropriate for RT-qPCR.

Described in Scientific Reports, the buffer was comparable to a commercially available reagent from Bio-Rad. The study also showed the buffer need not contain RNase inhibitor to be effective, and suggested it may be useful for high-throughput analyses.

"Our buffer is surprisingly simple," Haruhiko Murata, principal investigator on the study, told PCR Insider. "It's a Tris buffer with a little bit of sodium chloride, a little bit of a non-ionic detergent, and that's really all it is." The detergent used was Igepal CA-630 from Sigma, described as "chemically indistinguishable from Nonidet P-40, which is no longer commercially available," on that vendor's website.

Murata said he believes using crude lysate for RT-qPCR is becoming an increasingly popular strategy. It bypasses many of the rate-limiting sample prep steps. He attributed this trend to growing awareness among his colleagues of commercial cell lysis reagents. However, "Commercial reagents are great, but we were always a little bit uncomfortable that it's a black box" in terms of proprietary buffer recipes, he said.

In Murata's study, the commercial reagent was also associated with a significant price tag. The study estimated a cost per experiment of $1 per well for a 96-well plate-based RT-qPCR assay, with 60 percent of that cost coming from the commercial cell lysis buffer.

Thus, Murata's lab experimented with different recipes for in-house cell lysis buffers, and tested them against Bio-Rad's iScript Sample Preparation Reagent, a product launched in 2009. The study noted that there are other commercial products, such as Ambion Cells-to-CT and Invitrogen CellsDirect (both now offered by Thermo Fisher Scientific's Life Technologies); and Roche RealTime Ready Cell Lysis, but the researchers did not explicitly test these.

To test the buffers, the researchers used a previously developed microneutralization assay, in which MDCK-London cells in 96-well plates are infected with influenza virus and the infection is allowed to proceed. "Because we're using an RT-qPCR-based method, we're able to make our measurements at only six hours post-infection," Murata said.

At this point, the researchers remove the culture medium from infected cells, add lysis buffer, incubate a few minutes at room temperature, and the resulting crude lysate is then directly assayed. The RT-qPCR targets the matrix gene of the influenza virus, which results in a measure of viral replication. At the Office of Vaccines Research and Review, Murata's group uses this assay to test antibodies inhibiting viral replication, but he envisions it could also be used for screening chemical libraries.

The study tested varying concentrations of sodium chloride and detergent in the in-house cell lysis buffer, and measured copy numbers obtained via a one-step SYBR Green RT-qPCR with primers targeting the influenza matrix gene. It also subjected the crude lysates to various stresses, including frozen storage, freeze-thaw cycles, and incubation at 37°C for up to four hours. In addition, it combined some of these conditions to model routine handling scenarios in a typical lab. Different conditions had different impacts on RNA quality and RT-qPCR results, but the simple buffer showed similar results to the commercial buffer, even without added RNase inhibitor.

This was notable to Murata. "It's ingrained in anybody that works with RNA, you've got to really be careful; RNA is very labile and could degrade," he said. "We undertook this really prepared to add RNase inhibitor [to the buffer], but when we prepared our samples with this very simple buffer that doesn't contain RNase inhibitor, and then did these stress experiments, we found that the RNA is surprisingly stable."

"To us it was significant," Murata said. "If you were to add RNase inhibitor from a conventional commercial source ... to the concentration that most suppliers recommend, it's actually more expensive than commercial cell lysis reagents." The study calculated a cost of $3 per 100 microliters of buffer if RNase inhibitor were added to recommended concentrations.

Without this ingredient, the cost of Murata's cell lysis buffer is "negligible." The list price for 50 ml of Igepal CA-630 is $39, according to the Sigma website, and the lysis buffer uses a concentration of only 0.25 percent.

Murata said his lab is now trying to ascertain the mechanisms making the RNA so surprisingly stable using this buffer. He hypothesized that endogenous RNase inhibitor is also being released from the cells.

In addition to RT-qPCR, the study also showed "before and after" micrographs of cells treated with the cell lysis buffer versus iScript. This was to demonstrate that the lysis procedures were equivalent, and quite gentle, Murata said. "The overall gross architecture of the monolayer is intact, and you can see that the cell nuclei stay in place. If [nuclear contents] got released into your sample too, that might be problematic in certain contexts, if you're measuring a cellular gene that might actually lead to variability in your readings."

For smaller assays, the cost-benefit of using crude lysates may be different, Murata said. "If you're not interested in throughput ... it's probably always preferable to purify the RNA from your sample and then do your real-time PCR. That's the gold standard way to do it," he said.

He added, however, that for high throughput, "we think that this cell lysis approach in general, either using commercial reagents or using this in-house simple buffer that we describe, is a feasible approach." And, Murata said, since "as a quantitative method, real-time PCR is really hard to beat," being able to quickly, inexpensively, and accurately assay inhibitors of viral replication could be extremely useful.

Murata said his group is now testing whether their results are generalizable to other cells and gene targets. They are also doing experiments to determine what is making the RNA so stable in their assay. "It's nice to understand the mechanism; otherwise it's another black box. ... Knowing the mechanism, I think will also invite other people to try it."

Murata says he drew some inspiration from a review of RNA isolation methods done by John Chirgwin in the late 1980s. "He's really one of the pioneers that developed various RNA purification methodologies," Murata explained.

In an email to PCR Insider, Chirgwin, now a professor of medicine at Indiana University, said Murata's paper "looks good and carefully done." He suggested using crude lysate for PCR might be more ideal for cultured cells. Chirgwin himself now works on cancer biology using a model system that may not be amenable to this approach. "Most of the time my lab is isolating RNA from human tumor cells growing in mouse bone, which are full of all sorts of junk, requiring extensive purification before starting the PCR." However, Chirgwin added, "if we were doing only cell line PCRs, I would certainly give this a try. Fewer steps and less reagents are always good."

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