In order to test the success and reproducibility of the fingerstick method of RNA isolation we took two fingerstick capillary samples from 5 donors on two separate days (four samples total from each donor, 20 total samples). From each collection time point we chose one purified total RNA sample from each donor and went forward with the RNA amplification, fragmentation and hybridization protocols. A venous sample was also collected in parallel from each of the donors by a trained phlebotomist and processed according to standard protocols to represent the established method http://www.scripps.edu/researchservices/dna_array
]. All samples were assayed on Affymetrix HGU133 2.0 GeneChips using the Nugen standard array protocol for cDNA hybridization (Nugen FL-Ovation™ cDNA Biotin Module V2 - now called the Encore™ Biotin Module - user guide, http://www.nugeninc.com/tasks/sites/nugen/assets/File/user_guides/userguide_encore_biotin.pdf
Sample Collection, RNA Isolation, and Purification - Fingerstick Method
We followed the PAXgene Blood RNA kit (product# 762164) protocol for RNA isolation and purification, with the exception of one modification. After the first spin, we washed the pellet with 1 mL RNase free water instead of 4 mL due to its small volume. We initially tested a "scaled down" version of the entire PAXgene protocol, but, through further testing, we found that using the standard volumes of buffers and washes had no effect on the yields and were easier to employ (data not shown). Furthermore, we also found the DNase step in the protocol was crucial for the yield and fidelity of the amplified cDNA. Without the DNase step, contaminating DNA was subsequently amplified causing the GAPDH and Actin ratios used as quality control metrics on the GeneChips to be abnormally high (data not shown).
RNA Yield, Purity and Integrity - Fingerstick Method
From 20 samples of 70 μL fingerstick blood, the average total RNA yields ranged from 138 to 430 ng (average of 255.7 ± 72.6 ng) which was well above the maximum of 50 ng required for the Nugen Ovation RNA Amplification System v2 (Nugen, San Carlos, CA). While we did experience higher than normal OD260/280
ratios, an average of 3.9 ± 1.35, and lower OD260/230
ratios, an average of 0.09 ± .07, we found that this did not affect downstream applications and was probably caused by the high concentration of salts in the elution buffer relative to the low concentration of RNA http://www.flychip.org.uk/protocols/gene_expression/rna_qc.php
]. To assess the quality of the RNA, the samples were then run on the Agilent 2100 BioAnalyzer using an RNA PicoChip. Due to sample anomalies identified by the software, two of the 20 samples were unable to be assigned a RNA Integrity Number (RIN); however, the traces looked normal and this appeared to have no affect on downstream applications for these two samples. The RIN numbers of the RNA from the remaining 18 samples were between 8.9 and 9.9 (average of 9.3 ± 0.25), which indicates RNA of high quality and integrity [19
RNA Yield, Purity and Integrity - Venipuncture Method
The total RNA from the 5 normal venipuncture PAXgene blood collection tubes was extracted and purified according to the PAXgene Blood RNA kit (product# 762164) protocol. From 2.5 mL of blood the yield ranged from 4.1 to 7.9 μg of total RNA (average 5.96 ± 1.5 μg) with an average OD260/280ratio of 2.0 ± 0.03 and an average OD260/230 ratio of 1.0 ± 0.45. To assess the quality and integrity of the RNA, the samples were then run on the Agilent 2100 BioAnalyzer using an RNA NanoChip. The average RIN of the 5 total RNA samples obtained by venipuncture was 9.2 ± 0.09.
RNA Amplification, Labeling, and Fragmentation
50 ng total RNA from each donor was taken from all three sample sets (fingerstick 1, fingerstick 2, and venipuncture) and subsequently amplified using the Nugen Ovation® RNA Amplification System V2 (Cat.# 3100) and Ovation® WB Reagent (Cat.# 1300). The total RNA yields and Agilent traces for these 15 samples are shown in Figure . After amplification, the samples were purified according to the Nugen user guide instructions using the Qiagen QIAquick® PCR Purification Kit (Qiagen, Cat. #28104). As shown in Figure , the two batches of fingerstick samples (10 total) had cDNA yields ranging from 4.7 to 7.9 μg (average of 7.0 ± 0.88 μg), all satisfying the minimum requirement of 4.4 μg cDNA needed for Affymetrix chipping. The average OD260/280ratio of the 10 samples was 1.9 ± 0.01.
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 (more ...)
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 (more ...)
For the venipuncture samples (5 total), the cDNA yields ranged from 8.2 to 9.3 μg (average of 8.6 ± 0.43 μg) (Figure ) with an average OD260/280
ratio of 1.9 ± 0.01. Before fragmenting and labeling the cDNA with the Nugen FL-Ovation™ cDNA Biotin Module V2 (Cat.# 4200), the BioAnalyzer was used again to determine quality of the amplified whole cDNA (Figure ) and the results showed that all three sample sets had a consistent profile and size distribution across the RNA input range, indicating cDNA of good quality for array hybridization [Nugen Ovation RNA Amplification System v2 Technical Report #1, http://www.nugeninc.com/tasks/sites/nugen/assets/File/technical_documents/techdoc_ov_ampv2_rep_01.pdf
cDNA Hybridization and GeneChip Processing
GeneChip Data Quality Control
The Affymetrix quality metrics for the fingerstick samples are given in Table and all the Venipuncture samples in Table . Both the fingerstick and the venous samples had similar average background (30.8 vs. 29.5) and scale factor (2.7 vs. 2.8). The average % present calls were slightly higher for the venipuncture samples (56% vs. 52.5%). The fingerstick samples had higher 3'/5' GAPDH and β-Actin ratios on average.
Affymetrix quality metrics for all fingerstick samples.
Affymetrix quality metrics for all venipuncture samples.
Correlation Coefficients for Fingerstick and Venipuncture Samples
We calculated correlation coefficients for each donor comparing fingerstick collection 1 vs. fingerstick collection 2 and also each fingerstick collection vs. the venipuncture collection (Table ). Both fingerstick collections were highly correlated with r2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r2 values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.93 to 0.96 for fingerstick collection 2.
r2 correlation values for fingerstick and venipuncture samples
GeneChip Present/Absent Call Analysis
We analyzed the degree of disagreement in Affymetrix present/absent calls between the fingerstick collections and also between the fingerstick and venous collections for each donor. Disagreement is described as a change in the present, marginal or absent calls between any two comparisons. Within each comparison we binned the average signal intensities of each probe set in the ranges between 0-100, 101-250, 251-500, 501-1000 and >1001. We got similar results for all three comparisons that we performed. There was an inverse correlation between the signal intensities and the disagreement calls for the probesets in each bin (Figure &). In contrast, the number of probesets in agreement was not affected by the signal intensities irrespective of their bins, except at the lowest signal intensities (0-100).
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 (more ...)
We further analyzed the disagreement as the number of calls that changed from present to absent and vice versa to test the hypothesis that a change from absent to present would indicate higher sensitivity for the method used. On average, there was a higher number of disagreement calls, namely, change in call of a probe set from present in the venipuncture method to absent in the fingerstick method. However, there were also a number of calls that changed from present in the fingerstick collection to absent in the venipuncture collection (Table ). For example, Donor 2 changed calls from present, in the venipuncture method, to absent, in the fingerstick method, at an average rate of 6.7%. On the other hand, Donor 2 changed calls from absent, in the venipuncture method, to present, in the fingerstick method, at an average rate of 4%.
An analysis of the absolute change and percent change in calls between the fingerstick and venipuncture collections.