Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of beta-tubulin deactylase

10.1093/nar/gkw1143NUCLEIC ACIDS RESEARCH45

Remarkable stability of mRNA and miRNA at repeated freeze-thaw cycles in the <i>in-house</i> lysates.

<p>M14 cultured in 96-wells at 10⁴ cells per well (biological triplicates) were directly lysed by (A) in-house or (B) Bio-Rad lysis buffers. Cell lysates were collected and aliquoted. The lysates were then frozen at −70°C, thawed in a temperature-controlled water bath at 25°C, and the freeze-thaw cycles repeated. The miRNA and mRNA expressions were quantified following 1, 3, 5, 10 freeze-thaw cycles. The qPCR assays from set 2 were used for quantifications. Ct was plotted against freeze-thaw cycles to examine the stability of RNA. The experiments were conducted in biological duplicates.</p

Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

<div><p>Substantial efforts have been devoted to <i>in vitro</i> testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs) are known to regulate most cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from cell lysates (cell-to-Ct). Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Quantification of RNA species using lysates prepared from an <i>in-house</i> and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the cell lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.</p></div

Extraction variability in mRNA/miRNA detection.

<p>Box plot representation of the coefficient of variation (CV) calculated from the Ct values for (A) mRNA (RPS2 and VDAC1) and (B) miRNA (hsa-miR-103, hsa-miR-532-3p, and hsa-miR-485-5p). Each box represented CVs of the combination of mRNAs/miRNAs detected from 7 cell lines. The 25th percentile to the 75th percentile (boxes), and ranges (whiskers) were shown. Significant differences in CVs between Trizol reagent and the cell-to-Ct reagents were calculated using the unpaired, two tailed student's t-test. *, p<0.05; **, p<0.005. Primer design, assay efficiency and intra- and inter-assay variations were reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072463#pone.0072463.s005" target="_blank">Table S1</a>.</p

Excellent linearity of RNA detection.

<p>M14 cell line cultured in 96-wells at various densities (10, 100, 1000, 10,000 and 100,000 cells per well) were lysed directly by (A) in-house or (B) Bio-Rad lysis buffer. The cDNA samples generated from cell lysates were amplified by PCR assays from set 2. Standard curves for isolated RNAs were plotted as Ct versus Log (cells per RT). The experiments were conducted with biological duplicates.</p

Extraction efficiency in the detection of mRNA and miRNA.^a

a<p>The detected Ct values of the miRNA and mRNA assays from samples prepared with cell-to-Ct reagents were compared against Trizol reagent. The average ΔCt (Ct of cell-to-Ct reagents – Ct of Trizol) ± S.E.M. of the biological quadruplicates are summarized in the table. The values in bold represents average ΔCt >2 cycles.</p

Validation of extraction variability and efficiency.

<p>M14 cell line (10⁴ cells) was cultured and lysed with the Trizol reagent, in-house and Bio-Rad cell-to-Ct reagents. After reverse transcription, the cDNA samples were amplified by RPS8, ZNF706, RNF167, hsa-miR-29a, hsa-miR-197, hsa-miR-297 PCR assays. Graph bars represent (A) CVs and (B) detected Ct values of the biological quadruplicates. The error bars referred to the S.E.M and the significant differences in CVs between the Trizol reagent and cell-to-Ct reagents were calculated using unpaired, two tailed student's t-test. *, p<0.05; **, p<0.005.</p