A team from the University of Gothenburg working with Life Technologies has published a technique combining reverse transcription and proximity ligation to allow quantitative PCR measurements of the DNA, RNA, and proteins from a single cell.
The approach, described online in Clinical Chemistry last month, requires only a single standard real-time PCR instrument and yields the same qPCR output for all analytes, thus allowing the possibility of comparative analyses and correlation studies of DNA expression, transcripts, and protein levels at the single-cell level.
Anders Ståhlberg, the study's first author, told PCR Insider this week that the technique is one of the first to offer a way to correlate different types of analytes. "You can see the delay between DNA transcription, to translation, and what happens when you manipulate this," he said.
In an editorial discussing the paper also published in Clinical Chemistry this month, researchers from SciLifeLab in Uppsala, Sweden — the group that developed the proximity ligation, or PLA, method — wrote that new approaches like that of Ståhlberg and his colleagues "illustrate the growing opportunity to characterize critical cellular functions more fully at the nucleic acid and protein levels."
The Uppsala researchers' PLA technique has been commercialized through Olink Bioscience, which has also licensed the platform to Life Technologies.
PLA uses pairs of antibodies attached to unique oligonucleotides. When the antibodies bind to their target protein, the attached DNA strands come into close proximity, causing them to ligate and forming a new DNA amplicon that can then be quantified using real-time PCR or next-generation sequencing.
Ståhlberg said he has been working with Life Technologies on their applications of PLA, and that the company has also filed for a patent on the lysis method his group used in the combined approach described in the new paper.
According to Ståhlberg, other groups have combined PLA and qPCR for DNA and RNA detection, but not at the single-cell level.
"This is because when you run these [PLA] assays, you usually get a background signal. We were able to [reduce that] background to push that to very few molecules," he said. "We cannot say we have one molecule, but maybe we can say we have 20 or 100 in a cell and that's enough for high to medium expressed proteins."
Ståhlberg said his team approached building the combined single-cell method by first establishing a cell-sorting and lysis approach that would be amenable to DNA, RNA, and protein analysis.
"The first thing was that we needed to make all these approaches work with the same kind of experimental setup, and by that I mean the lysis," he explained.
Since PLA and RNA/DNA analyses require different reaction conditions, the group needed to begin the method by splitting cells into fractions, Ståhlberg said.
"Normally, you have different lysis conditions for DNA and proteins – so we came up with a new lysis strategy, which uses a quite gentle lysis buffer – but we spent a lot of time working to have the lysis buffer be equal or better [than previous methods] and suitable for both protein and DNA and RNA."
As described in the paper, Ståhlberg and his partners tested the method using transiently transfected human cells from a fibrosarcoma cell line. The group lysed cells using a gently analyte-releasing buffer method, and then proceeded to parallel workflows for DNA, RNA, and proteins using Life Technologies PLA protocols and TaqMan assays.
The group wrote that all of the assays had high analytical sensitivity and dynamic range, and all were able to detect their respective target molecules at the single-cell level.
"The sensitivity in principle with RNA and DNA is down to one molecule," Ståhlberg said. "With the protein analysis, the main limitation is how good the antibodies you have are. It's hard to count the number of proteins, because you always have this background signal, so …we say we have this number or higher. It's relative quantification. [In the paper] we show we can do it but we don't stress the idea of how many molecules because that depends a lot on what antibodies you use."
Ståhlberg said that the group is most excited about the approach because it offers the same readout for all target analytes. "If you use different techniques you get very different readouts. But here you get a qPCR readout for protein, DNA, RNA, miRNA."
"I think that’s very appealing for people, that you get the same kind of parameter out. You don't need any advanced software – you can use your standard qPCR software," he said.
Ståhlberg said his group is planning to use the method to study the interplay of oncogenes and transcription factors. "This is important for tumor cell proliferation. We want to look at downstream direct targets when you have a lot of the protein or a little," he said.
In their recent commentary on the paper, the Uppsala researchers note that Ståhlberg's team has only demonstrated the combined approach in a "somewhat artificial system," in focusing on a transfected plasmid and its associated transcript and fusion protein rather than unmodified cell DNA, RNA, and proteins.
Additionally, the commenters suggested that the multiple-analyte approach may be applicable beyond real-time PCR. "It is easy to see that the described approach can be generalized to obtain a higher throughput and a more complete understanding … [using] next-generation sequencing to read out DNA, RNA, and protein concentrations," the authors wrote.