Quantitative analysis of mRNA amplification by in vitro transcription

Abstract

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Figure 1

Minimizing primer concentration and RT reaction volume reduces the production of template-independent in vitro transcription product. Products from control, standard and optimized in vitro transcription amplification reactions resolved by (A) native and (B) denaturing agarose gel electrophoresis and stained with SYBR Gold. (A) Lane 1, 50 ng RNA ladder; lane 2, 100 ng poly(A) RNA; lane 3, 5% in vitro transcription reaction containing no template but 500 ng of (dT)-T7 primer; lane 4, 10% no template control standard amplification reaction using 500 ng primer; lane 5, 10% standard amplification reaction using 10 ng total RNA and 500 ng primer; lane 6, 100% no template control optimized amplification reaction using 10 ng primer; lane 7, 100% optimized amplification reaction using 10 ng total RNA and 10 ng primer. Note the lack of template-independent product in lane 6 and the appropriate mobility of the amplification products in lane 7 as compared to lanes 4 and 5. (B) Lane 1, RNA ladder; lane 2, identical reaction product in (A) lane 3; lanes 3 and 4, product of single round of amplification from 10 µg and 200 ng total RNA; lanes 5, 6 and 7, products from two rounds of amplification from 10, 2 and 0 ng total RNA. An equal fraction of each product was loaded in lanes 6 and 7.

Figure 2

Single-stranded nucleic acid-binding protein enhances processivity of RT. Alkaline gel electrophoresis of cDNA product from RT reactions containing increasing amounts of T4gp32 single-stranded binding protein. Lane 1, no T4gp32; lane 2, 14 µg/ml T4gp32; lane 3, 200 µg/ml T4gp32. Note the reduction in the inter-band smear and secondary bands in lanes 2 and 3.

Figure 3

Scatter plots of gene frequencies from replicate hybridizations and replicate amplification reactions. (A) Scatter plot showing the reproducibility of the Affymetrix GeneChip platform: a single amplification product from 10 µg total RNA was hybridized to multiple chips. (B–E) Scatter plots showing the reproducibility of amplification: gene frequencies from replicate amplification reactions using decreasing amounts of starting total RNA. (B) 10 µg, (C) 200 ng, (D) 10 ng and (F) 2 ng. (E) The complete set of correlation coefficients for all replicates performed.

Figure 4

Scatter plots of gene frequencies comparing increasing amounts of amplification. The scatter plots compare the average gene frequency from four independent amplification/labeling reactions using 10 µg total RNA to the gene frequencies from amplification of the same total RNA diluted to (A) 200 ng, (B) 10 ng and (C) 2 ng. (D) The complete set of correlation coefficients including additional comparisons.

Figure 5

Scatter plots of gene frequencies for diverse RNA samples. The scatter plots compare the gene frequencies of embryonic versus adult RNA after amplification from different starting amounts of total RNA: (A) 10 µg, (B) 200 ng and (C) 10 ng total RNA. (D) The complete set of correlation coefficients.

Figure 6

Scatter plots of t scores for the differences in observed gene frequency between two diverse RNA samples with and without amplification. t scores were calculated from the data shown in Figure 5. (A) Control t scores from separate pairs of amplification reactions using 10 µg starting total RNA (n = 2). Comparison of t scores derived from 10 µg data (n = 4) and data from amplification from (B) 200 ng (n = 2) and (C) 10 ng (n = 2) total RNA. (D) Overlap between lists of the most significantly different gene frequencies in each data set (highest absolute t scores). *In the intersection between 10 µg and 10 ng data sets, nine genes from each top 20 list do not intersect, but the most discrepant of those 18 genes is found in the top 80 of the other data set.