Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

Abstract

Background: Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 microl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit.

Results: From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r(2) values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r(2) values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.

Conclusions: Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.

Figure 1

Fingerstick and venipuncture RNA yields and Agilent 2100 bioanalyzer traces including RNA integrity numbers (RINs). (A) Fingerstick starting material: 70 uL, venipuncture starting material: 2.5 mL, yields normalized to 70 uL. (B, C, D) Agilent 2100 Bioanalyzer total RNA traces using the PicoChip (B & C) and the NanoChip (D).

Figure 2

Fingerstick and venipuncture cDNA yields and Agilent 2100 Bioanalyzer traces. The samples included in this figure refer to the same samples as Figure 1. (A) Yields of cDNA post Nugen Ovation amplification of 50 ng total RNA. Yields of cDNA from both fingerstick and venipuncture samples were always above the 4.4 ug cut-off needed to continue with hybridization to GeneChip (B, C, D) Agilent 2100 Bioanalyzer cDNA traces using the NanoChip.

Figure 3

Inverse correlations between signal intensities and number of disagreement calls. The figures A, B, and C illustrate the inverse correlation between signal intensities and the number of disagreement calls; as the signal intensity increases, the disagreement calls between fingerstick samples and venipuncture samples drastically decreases.