Example 1.High-quality cDNA can be obtained from cell lysates.

The optimized lysis solution efficiently inhibits RNA degradation during treatment. RNA in the lysate is stable on ice for at least 2 h. High-quality cDNA can be synthesized using highly efficient reverse transcriptase "ReverTra Ace™" with low contamination of genomic DNA because of preceding DNase I treatment. The reverse transcriptase is supplied as a master mix reagent containing optimally mixed primers (random and oligo dT) to achieve effective cDNA synthesis.

Comparison of the efficiency with purified RNA

SuperPrep™ Cell Lysis & RT Kit for qPCR (Code No SCQ-101) synthesized cDNA using the lysate from 2.5×104 HEK293 cells in a 40 µL reaction. cDNA was synthesized using 66.6 ng of total RNA, corresponding to 2.5×104 HEK293 cells, using a cDNA synthesis kit (ReverTra Ace™ qPCR RT Master Mix [Code No. FSQ-201]) in a 40 µL reaction. Fifteen housekeeping genes were then analyzed by SYBR^® Green real-time PCR using the synthesized cDNA. High correlation was observed between the two methods. The method using SuperPrep™ facilitated the expression analysis by real-time PCR without time-consuming RNA purification steps.

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Evalution of efficiency for expression analysis of cells with high RNase activity

Expression analysis using a lysate from U937 cells is difficult because of high RNase activity. In this experiment, the lysates (8 µL) from 1×10⁴, 1×10³, 1×10² and 1×10¹ cells were used. β-actin genes were analyzed by SYBR^® Green real-time PCR assay using cDNA synthesized from the lysates in 40 µL reaction. The same experiments were performed using two similar systems (SuperPrep™ and another company's product [Company A]) and conventional method using corresponding amounts of RNAs. SuperPrep™ showed successful amplification, comparable to the conventional method.

Example 2.Stability test of the cell lysates.

cDNA were synthesized from lysates that had been left on ice for 0–24 h after lysing of 4×10⁴ HeLa and U937 cells using SuperPrep™. β-actin genes were detected using TaqMan™ real-time PCR assay with THUNDERBIRD™ Probe qPCR Mix (Code No. QPS-101). The results were compared with that from the other company’s system (Company A). The results suggest that the RNA in the cell lysates is stable for at least 2 h. Lysates from U937 cells showed higher RNase activity than the other cells and tended to deteriorate in storage over 2 h.

NOTE:

RNase activity depends on the type and number of cells. The cell lysates should be placed on ice after preparation and cDNA should be synthesized immediately after preparing the lysates to minimize RNA degradation.

Example 3.Various real-time PCR reagents can be applied.

The synthesis cDNA can be used in various real-time PCR assay (TaqMan™ probe, SYBR^® Green etc.).
In addition, the cell lysate can be applied to one-step real-time PCR reagents.

TaqMan™ Probe assay using cDNA synthesized from cell lysates prepared by SuperPrep™

cDNAs were synthesized using the cell lysates (8 µL) prepared from 7.5×10⁴, 5×10⁴, 1×10⁴, 1×10³, 1x10² and 1×10] HeLa S3 cells by SuperPrep™ in 40 µL reaction. β-actin genes were detected by various real-time PCR reagents with TaqMan™ real-time PCR assay. Successful amplifications were obtained from all reagents tested and the THUNDERBIRD™ Probe qPCR Mix tended to show a better Ct than the other tested methods.

Example 4.Evaluation of the assay variation

HeLa S3 cells were incubated with or without 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h after seeding at 2×10⁴ cells/well in a 96-well culture plate. cDNA were synthesized from the lysates prepared from the cells washed with PBS(-). IL-6, IL-1β and β-actin genes were detected by TaqMan™ real-time PCR assay with THUNDERBIRD™ Probe qPCR Mix (Code No. QPS-101). After compensation of the Cts of IL-6 and IL-1β by that of β-actin, the ΔΔCt between with or without PMA and Z' factors* were calculated.
Z' factors from SuperPrep™ were superior to that from the other system (Company A).

*The Z' factor is a simple statistical parameter that is used to assess the quality of high-throughput screening (HTS) assays. A Z' score of ≥0.5 is generally considered to indicate good quality Z' can be calculated by the following formula.

Z'= 1-3 × [Δ Ct(+) standard deviation + Δ Ct(-) standard deviation]/| Δ Δ Ct|