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MessageBOOSTER™ Kit RNA Amplification and cDNA Synthesis for qRT-PCR Directly from a Single Cell without RNA Purification Judith E. Meis, Anupama Khanna, and Jim Pease, EPICENTRE Biotechnologies Introduction Quantitative reverse-transcription PCR (qRT-PCR) gene expression studies from very small number of cells (e.g., 1-1,000 cells) can be challenging for a variety of reasons: • • • • • Difficulty in purifying extremely small amounts of total RNA. A limited number of reactions can be performed, and very few transcripts analyzed, per sample. Lack of sensitivity, especially when detecting low- and medium-abundance messages. The need to collect samples often. The inability to archive samples for future use. The new MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit eliminates these difficulties by enabling RNA amplification and cDNA synthesis directly from cell lysates, without the need to purify total RNA. Here we demonstrate that a MessageBOOSTER Kit reaction produces enough cDNA directly from the lysate of a single cell for thousands of qPCRs, and that the cDNA produced enables sensitive detection of even low-abundance transcripts. Methods An overview of the process for the MessageBOOSTER cDNA Synthesis from Cell Lysates Kit is illustrated in Fig. 1. Briefly, cells are lysed and the poly(A) RNA (mRNA) in the crude, whole-cell lysate is amplified by in vitro transcription. The amplified RNA is converted to cDNA, and the cDNA diluted up to 1,000-fold for qPCR. The single-day reaction will efficiently produce cDNA directly from lysates of 1 to 1,000 cells. Cell collection and lysis Cultured normal rat kidney (NRK) or HeLa cells were collected, counted, and placed into sterile 0.5-ml microcentrifuge tubes on ice. The cells were pelleted at 4°C at 500-1,200 x g for 5 minutes, and the culture medium was carefully removed. The cells were resuspended in 3 µl of QuickExtract™ RNA Extraction Solution (provided in the kit; see also p. 14) and lysed either by: i) freezing in liquid nitrogen and then thawing for 2 minutes at room temperature; or ii) vigorous vortex resuspension for 1 minute. The cell lysates were placed on ice. RNA amplification and cDNA synthesis The MessageBOOSTER Kit reaction was performed directly in the cell lysates. As shown in Fig. 1, an oligo(dT) primer containing a T7 promoter and MMLV Reverse Transcriptase were used to synthesize first-strand cDNA from poly(A) RNA. After second-strand cDNA synthesis, a high-yield in vitro transcription reaction was used to amplify the poly(A) RNA (mRNA). The in vitro transcription reaction generates thousands of RNA molecules for every molecule of double-strand (ds) 10 www.EpiBio.com cDNA template, and thus serves as a powerful engine for amplifying the poly(A) RNA. Following in vitro transcription, the samples were treated with DNase I to remove the ds cDNA template, as well as a large portion of genomic DNA. The amplified RNA was purified using an RNA Clean & Concentrator™-5 column (Zymo Research) and then reverse-transcribed, using random primers, into cDNA in a final volume of 12.5 µl. cDNA produced by the MessageBOOSTER Kit reaction was designated as “MessageBOOSTER cDNA.” Control reactions cDNA was synthesized directly from cell lysates without an RNA amplification step. This cDNA synthesis process, designated as the “cDNA-from-cells” procedure, was performed by reverse transcription using an oligo(dT) primer in a 20-µl reaction. In some cases, a commercial cDNA-from-cells–type kit was used. A MessageBOOSTER Kit reaction was performed without the initial reverse transcriptase. This control, designated as the “No-RT” control, was performed in order to evaluate the level of residual genomic DNA in the samples after the MessageBOOSTER Kit reaction. Real-time PCR The cDNA produced by the MessageBOOSTER Kit reactions was diluted 1:10 and 1:1,000 in water for subsequent qPCR. A 1-µl aliquot of the diluted cDNA was used for each 25-µl qPCR. qPCR was performed using the TAQurate™ GREEN Real-Time PCR System (EPICENTRE). Volume 16-2 • June 2009 MessageBOOSTER™ Kit Results Benefit of the MessageBOOSTER RNA amplification step for small samples The MessageBOOSTER Kit uses a linear RNA amplification step that generates >1,000 RNA molecules from each molecule of poly(A) RNA in the cell lysate. In addition, the RNA amplification includes a DNase treatment step that significantly reduces the genomic DNA in the lysate. cDNA was produced from a single HeLa cell using the MessageBOOSTER kit and the cDNA-from-cells procedure (which does not include an RNA amplification step). qPCR was performed using 1 µl of undiluted cDNA produced by each reaction and a primer pair for the LDHA transcript. The No-RT control reaction was performed in order to assess the amount of residual genomic DNA present after a MessageBOOSTER kit reaction. As shown in Fig. 2A, the LDHA transcript was readily detected using cDNA produced Cultured Cells Cell Lysis Cell Lysis Solution Cell Lysate No RT 1 cell No RNA amplification T7-Oligo (dT) Primer TTTT T7 Cycle AAAA TTTT T7 AAAA TTTT T7 T7 Round 1 Second-Strand cDNA Synthesis B In Vitro Transcription UUUU Round 2 First-Strand cDNA Synthesis 5 cells –d(RFU)/dT Round 1 First-Strand cDNA Synthesis Relative Fluorescence Units A aRNA (cRNA) Random Hexamers T,°C UUUU AAAA RNase H AAAA cDNA PCR (provided by user) Figure 2. A MessageBOOSTER™ Kit reaction enables sensitive and specific detection of low-abundance transcripts. A, qPCR detection of the LDHA transcript in lysates from five HeLa cells (purple) and a single HeLa cell using cDNA produced by a MessageBOOSTER Kit reaction (blue), by a cDNA-fromcells procedure, without benefit of an RNA amplification step (green), and from a No-RT control reaction (black). B, Melt-curve analysis of the qPCR products revealed that the MessageBOOSTER cDNA produced an authentic LDHA amplicon, while the cDNA-from-cells procedure and No-RT control reaction generated nonspecific amplification products; these results were confirmed by gel electrophoresis (not shown). Figure 1. An overview of the procedure for the MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit. A kit reaction amplifies the poly(A) RNA (mRNA) directly from a crude cell lysate without the need for RNA purification. The amplified RNA is then reverse-transcribed to cDNA that can be diluted up to 1,000-fold for qPCR. Volume 16-2 • June 2009 www.EpiBio.com 11 Relative Fluorescence Units MessageBOOSTER™ Kit 1:100 1:1,000 1:10 undiluted NRK-cell lysate. Thus, a MessageBOOSTER Kit reaction can produce sufficient cDNA for >1,000 qPCRs from a single-cell lysate. The number of qPCRs that can be performed using cDNA produced by the MessageBOOSTER Kit is thus dependent on the number of cells and the abundance of the transcript(s) of interest. Conclusions Cycle Figure 3. A MessageBOOSTER™ Kit reaction produces sufficient cDNA from a single-cell lysate for thousands of sensitive qPCRs. qPCR was performed using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and 1:1,000 diluted (purple) cDNA from a lysate of a single NRK cell. The low-abundance PBGD transcript was readily detected. by a MessageBOOSTER Kit reaction from five cells (CT = 19) and from a single cell (CT = 22). The cDNA-from-cells and the No-RT control produced only nonspecific signals, as determined by the melting curve analysis in Fig. 2B. The melting temperature of the nonspecific products was significantly different from that of the LDHA-specific amplicon. Thus, the RNA amplification step, included in the MessageBOOSTER Kit reaction, enables sensitive and specific detection of transcripts from a single-cell lysate and reduces the amount of genomic DNA to undetectable levels. In contrast, the cDNAfrom-cells procedure yielded cDNA that did not detect the desired transcript in qPCR. Sensitive detection of low-abundance transcripts We further investigated the detection limit of a low-abundance transcript using cDNA produced by a MessageBOOSTER Kit reaction from a single-cell lysate. MessageBOOSTER cDNA, produced from a single NRK-cell lysate was diluted 1:10, 1:100, and 1:1,000, prior to qPCR. As shown in Fig. 3, cDNA diluted 1:1,000 enabled detection (CT = 29) of the low-abundance PBGD transcript. Melting curve analysis (data not shown) confirmed that the correct amplicon was produced. Thus, a MessageBOOSTER Kit reaction enables sensitive detection of a low-abundance transcript using up to a 1,000-fold dilution of the cDNA produced. The data presented demonstrate that cDNA produced by a MessageBOOSTER cDNA Synthesis from Cell Lysates Kit reaction eliminates the difficulties frequently encountered when performing qPCR gene expression studies from very small numbers of cells. We have shown that: • The kit enables the user to amplify the mRNA and convert it to cDNA directly from a cell lysate without the need to purify total RNA. RNA amplification and cDNA synthesis directly from cell lysates eliminates the difficulty and irreproducibility of purifying RNA from very small numbers of cells. Because the RNA amplification and cDNA synthesis reactions are performed directly in the cell lysates, there is no loss of RNA as can occur when purifying RNA from very small samples. • A MessageBOOSTER Kit reaction, which includes a linear RNA amplification step, significantly improves the sensitivity of detecting even low-abundance transcripts from as little as one cell. • A single MessageBOOSTER Kit reaction produces enough cDNA for thousands of qPCRs. More qPCRs per sample means that samples are collected less frequently and that there is plenty of cDNA produced to enable the user to archive the sample for future use. Cat. # Quantity MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit MBCL90310 10 Reactions Are you performing qRT-PCR studies using purified total RNA? Try the original MessageBOOSTER™ cDNA Synthesis Kit for qPCR (Catalog Numbers MB060110, MB060124) which enables hundreds of sensitive qRT-PCRs from as little as 10 pg of purified total RNA. Visit www.EpiBio.com for details. Yield of amplified cDNA/number of qPCRs obtained The final step of a MessageBOOSTER Kit reaction converts amplified RNA to cDNA by reverse transcription in a reaction volume of 12-15 µl. As shown in Fig. 3, 1:10, 1:100, and 1:1,000 serial dilutions of the cDNA enabled sensitive detection of the low-abundance PGBD transcript from a single 12 www.EpiBio.com Volume 16-2 • June 2009