2 DNA Microarray Core, The Scripps Research

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1 Comparison of RNA, DNA, small RNA (including mirna) and protein yields from liver biopsy samples using two commercially available tissue preservation reagents: RNALater and AllProtect Terri Gelbart 1, Nina Gao 1, Tony S. Mondala 1,2, Sunil M. Kurian 1, Kim M. Olthoff 3, Michael Abecassis 4, Daniel R. Salomon 1 1 Department of Molecular and Experimental Medicine and 2 DNA Microarray Core, The Scripps Research Institute, La Jolla CA, 3 Hospital of the University of Pennsylvania, Philadelphia, PA, 4 The Northwestern University Feinberg School of Medicine, Chicago, IL Objective: Commercially available tissue reagents provide immediate stabilization of DNA, RNA, and protein in tissue samples. The most common tissue stabilization reagent is RNALater (Qiagen, Cat. No ), which we have been routinely using in our laboratory for preservation of RNA, DNA and protein from both cells and tissues. Tissue or cell pellets are mixed with RNALater and stabilized initially at room temperature for a few hours to allow the reagent to penetrate the cells and then can be stored at room temp for 1 week or stored at -80 o C indefinitely till used. A recent development in tissue stabilization has been the use of Allprotect Tissue Reagent (Qiagen, Cat No ), which provides immediate stabilization of DNA, RNA, and protein in tissue samples at room temperature. The claim is that this preserves the in vivo profile of DNA, RNA, and proteins, allowing reliable downstream analysis. Stabilized tissues can be transported at C for up to 7 days, or stored at 2 8 C for up to 12 months. For longer storage, stabilized tissues can be archived at 20 C or 80 C. In this study, we used 11 matched liver biopsy cores to compare the RNA, DNA, microrna and protein yields using these two tissue stabilization reagents. Methods: 11 liver biopsy cores in duplicate were collected and stored in tubes containing either RNALater or AllProtect and stored at -80 C over a range of 2 weeks to 2 months prior to shipping to Scripps for processing. Within one month of receiving, all samples were thawed on ice and processed immediately using the AllPrep

2 DNA/RNA/Protein Mini Kit (Qiagen, Cat. No ), that allows simultaneous purification of DNA, RNA, and protein from the same cell or tissue sample. Briefly, all biopsy cores were visually adjusted for size and were then homogenized with a VWR Mortor Mixer (VWR, Cat. No ) and a disposable pestle (VWR, Cat. No ). The homogenate was then passed through a 21 gauge needle and the lysate was processed as per the AllPrep manufacturer s instructions for the extraction of RNA, DNA, microrna and protein. Purification of mirna from cells and tissues was done using AllPrep DNA/RNA/Protein Mini Kit and RNeasy MinElute Cleanup Kit. The MinElute Cleanup Kit (Qiagen, Cat. No.74204) protocol purifies a small RNA fraction which contains mirna, trna, and other small RNA <200bp. RNA and small RNA s were quantified on a NanoDrop 1000 and the quality analyzed on an Agilent Bioanalyzer DNA samples were quantified on a NanoDrop Protein was dissolved using 0.4% RapiGest in 100mM TRIS (ph 8.0) and quantified using the BCA Protein Assay Kit (Pierce). Results: Table 1 shows the comparison of the RNA yields from the samples stored in the RNALater and the AllProtect reagents. The average RNA yields from biopsies stored in RNALater was lower than those obtained from the AllProtect (110ng/µl vs. 162ng/µl; p<0.06). However, the RNALater samples had a superior RNA Integrity Number (RIN) compared to the AllProtect samples (8.7 vs. 8.1; p<0.003). RIN is a software tool designed to help scientists estimate the integrity of total RNA samples. Using this tool, sample integrity is no longer determined by the ratio of the ribosomal bands, but by the entire electrophoretic trace of the RNA sample. This includes the presence or absence of degradation products. Figure 1 shows a representative Agilent electropherogram for a few of the RNAs extracted in the study (Lanes 1-10). Table 2 shows the comparison of the DNA yields from the samples stored in the RNALater and the AllProtect reagents. The average DNA yields from biopsies stored in RNALater did not differ from those obtained from the AllProtect (72ng/µl vs. 80ng/µl). Table 3 shows the comparison of the small RNA yields (including the mirna fraction) from the samples stored in the RNALater and the AllProtect reagents. The average small RNA yields from biopsies stored in RNALater were slightly lower when

3 compared to those obtained from the AllProtect (128ng/µl vs. 155ng/µl). However this was not statistically significant. Figure 1 shows a representative Agilent electropherogram for two of the small RNAs extracted in the study (Lanes 11 & 12). Table 4 shows the comparison of the protein yields from the samples stored in the RNALater and the AllProtect reagents. The average protein yields from biopsies stored in RNALater were slightly lower when compared to from those obtained from the AllProtect (44µg vs. 49µg). However this was not statistically significant. This preliminary study reveals that there are no significant differences in the RNA, DNA, small RNA and protein quality and yields between the storage and stabilization reagents RNALater and AllProtect. We note a modest trend towards better yields of RNA using the AllProtect. Technical notes: From a technical standpoint the AllProtect is very viscous and is a little more difficult to work with since it required blotting each biopsy to remove the excess AllProtect. RNAlater does not require this step. But on the other hand the AllProtect freezes as a clear solution so the biopsy size can be assessed before thawing. Based on a per sample cost analysis, AllProtect is almost 5 times more expensive than RNALater. The only obvious advantage of AllProtect is that the samples can be stored initially at room temperature for almost a week which may make initial holding and shipping of samples easier in certain situations though freezers are readily available in most clinical settings.

4 Table 1: Comparison of the RNA yields from the samples stored in the RNALater and the AllProtect reagents

5 Table 2: Comparison of the DNA yields from the samples stored in the RNALater and the AllProtect reagents

6 Table 3: Comparison of the small RNA (including mirna) yields from the samples stored in the RNALater and the AllProtect reagents

7 Table 4: Comparison of the Protein yields from the samples stored in the RNALater and the AllProtect reagents

8 Figure 1: A representative Agilent electropherogram of a few of the RNAs (Lanes 1-10) Figure and 1 small RNAs (Lanes 11 & 12) extracted in the study. 28 and 18s RNA Small RNAs