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PCR Inhibition by Reverse Transcriptase Leads to an Overestimation of Amplification Efficiency Oleg Suslov and Dennis A

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PCR Inhibition by Reverse Transcriptase Leads to an Overestimation of Amplification Efficiency Oleg Suslov and Dennis A Published online November 27, 2005 Nucleic Acids Research, 2005, Vol. 33, No. 20 e181 doi:10.1093/nar/gni176 PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency Oleg Suslov and Dennis A. Steindler* McKnight Brain Institute of the University of Florida, FL, USA Received June 30, 2005; Revised September 19, 2005; Accepted October 25, 2005 ABSTRACT dilution will negatively affect the precise detection of rarely expressed genes. There are other approaches to reduce the RT This study addresses the problem of PCR inhibition impact on PCR: by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA (i) heating the RT reaction before PCR (3); is taken for RT reaction, and it is more visible for rarely (ii) introduction of T4 gene 32 protein (4); expressed transcripts. We show here that the inhibi- (iii) including of non-homologous RNA as a carrier (1); tion takes place regardless of what amount of tem- (iv) adding of foreign DNA (2); (v) excluding DDT from the RT reaction (8); plate is utilized for RT. The inhibition possesses a (vi) ethanol precipitating RT reaction (5); and global nature, i.e. the amplification of any given (vii) phenol extraction followed by alcohol precipitation of transcript may be compromised with different levels RT reaction (3). of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, The majority of studies of RT inhibitory effects, up until now, sometimes more than 100%, when the sequential were performed using regular PCR. In the present study, util- dilutions of unpurified RT sample are utilized to izing an organic extraction followed by ethanol precipitation build the calibration curve. The RT influences PCR of the RT reaction, and a real-time PCR approach, we were not only by inhibiting it. When microgram(s) of RNA able to perform a calculation of an approximate percentage of are taken for RT reaction, reverse transcriptase may inhibition in every dilution point of its unpurified counterpart RT reaction. A novel observation here is that a derived PCR cause overamplification of some transcripts under amplification efficiency of an unpurified sample may be incor- certain PCR conditions. The possible mechanism rectly assigned because of the effects of RT on PCR. It is of RT influence on PCR is presented, and a purifica- deemed important to remove reverse transcriptase before per- tion method is implemented to remove the effects of forming PCR. RT on PCR. MATERIALS AND METHODS INTRODUCTION Total RNA isolation Messenger RNA profiling relies heavily on a reverse transcrip- Total RNA was extracted from LN229 primary human glioma tion step which is accompanied by PCR. The recombinant cells, grown on six plates (50–70% of confluent) using the Moloney murine leukemia virus reverse transcriptase (RT), RNeasy Mini Kit (Qiagen) with DNase I treatment on the RNase H deficient (MMLV, HÀ) and avian myeloblastosis column. Eluted RNA was subjected to a second DNase I virus RT, RNase H deficient (AMV, HÀ) are the most fre- treatment in solution using Turbo DNA-free (Ambion). quently used reverse transcriptases for this purpose. There is After the treatment, total RNA was cleaned and concentrated a well-known inhibitory effect on PCR by RT components using phenol/chloroform/isoamyl alcohol (25:24:1), pH 5.2 (1–5). The introduction of micrograms of RNA into the RT (Amresco) followed by ethanol (EtOH) precipitation. Total step, followed by the extensive dilution of RT reaction before RNA was dissolved in RNase-free water (Ambion), and the PCR execution, is supposed to minimize this inhibition quality was confirmed using an HP 2100 Bioanalyzer (Agilent (1,3,4,6,7). These conditions often are not met for two reasons: Technologies). The readings gave a RIN (RNA Integrity Num- (i) there is not enough RNA available, and (ii) the extensive ber) value of 9. The isolated RNA had an A260/A280 ratio of 2. *To whom correspondence should be addressed. Tel: +1 352 392 0490; Fax: +1 352 846 0185; Email: [email protected] Correspondence may also be addressed to Oleg Suslov. Tel: +1 352 392 6754; Fax: +1 352 392 0025; Email: [email protected] Ó The Author 2005. Published by Oxford University Press. All rights reserved The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] e181 Nucleic Acids Research, 2005, Vol. 33, No. 20 PAGE 2 OF 12 The lack of phenol contamination was confirmed by measur- amount of either 50 ng or 1 mg of total RNA (LN 229) ing the absorbance at 270 and 275 nm using the UV spectro- were added. The total RNA dilution was made on yeast photometer, SmartSpec 3000 (BioRad). tRNA solution to introduce 100 ng of the carrier to the RT reaction. Each RT reaction was run in triplicate. The mixture DNA standards creation was heated at 65C for 5 min and incubated on ice for 3 min. Full-length transcripts of human beta-2-microglobulin (B2M) The first strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl and TATA box binding protein (TBP) were amplified by and 3 mM MgCl2), 5 mM DTT and 5 U of RNase Inhibitor PCR using proof-reading Platinum Taq DNA Polymerase (Invitrogen) were added into each tube. The tubes were incub- HiFi (Invitrogen). Polyadenylated mRNA fragment of plant ated at 42C for 5 min, then 200 U of SuperScript III (Invit- (Arabidopsis thaliana) lipid transfer protein 4 (LTP4)— rogen) were added and tubes were incubated at 50C for SpotReport mRNA spike 4 (Stratagene) was subjected to 40 min, 55C for 20 min and 70C for 15 min. Then, the SMART amplification (Clontech), following the manufac- cDNA:RNA hybrid was treated with 2 U of RNase H (Invit- turer’s instructions, except for the 50SMART oligonucleotide rogen) at 37C for 20 min. The dilution solution of a carrier which was modified to carry the T7 RNA Polymerase pro- (50 ng/ml yeast tRNA—41.5 ml) was added to each tube. The moter (T7-SWITCH primer). The full-length B2M, TBP and contents were mixed and split for two tubes; one tube was used LTP4 fragment were cloned into a pCR 2.1-TOPO vector for further phenol/chloroform/isoamyl alcohol (PCI) treatment (Invitrogen). The constructs were sequenced and used in a followed by ethanol precipitation (PCI sample), and the new PCR to produce the corresponding amplicons, which second tube received no further purification (Figure 1). were cleaned using a QIAquick PCR purification kit (Qiagen). The single band of amplification product was confirmed by Phenol/chloroform/isoamyl alcohol (PCI) procedure running a 1.5% agarose gel containing ethidium bromide for followed by ethanol precipitation visualization. PCI samples (31.2 ml) were subjected to organic extraction To generate standard calibration curves, 6-fold serial dilu- followed by EtOH precipitation. Linear Polyacrylamide tions of corresponding amplicons were made on 50 ng/ml yeast (LPA), GenElute LPA (Sigma), 15 mg(1mg/ml) was added tRNA as a carrier. The calibration curves have spanned to each sample before the PCI procedure, along with 1· TE, 4 orders of magnitude. The standards were kept at 4C. To pH 8.0 (Sigma) to bring the final volume to 100 ml. For extrac- check the reproducibility of the standard calibration curve, the tion of cDNA, 100 ml of PCI, pH 8.0 (Amresco) was added runs were carried out at least four times with the same standard to each PCI sample. Samples were then vortexed for 1 min, dilutions in a time period of over a month, on different days. allowed to stand for 5 min and then centrifuged at 7000 g for 5 min at room temperature. The procedure was carried out in RT reactions safe-lock tubes (Eppendorf). The aqueous phase was slowly For each reaction in a total volume of 20 ml, 500 ng oligo aspirated in a steady fashion and transferred into a new tube. (dT)(12–18), dNTP mix (0.5 mM each) and a corresponding Tris-EDTA, 1·, 100 ml was added to the extraction tube and Figure 1. Experimental layout: comparison of unpurified and PCI samples. PAGE 3 OF 12 Nucleic Acids Research, 2005, Vol. 33, No. 20 e181 the PCI procedure was repeated again, giving the final volume of 200 ml. We then added 1/10 vol of 5 M ammonium acetate (Ambion) and mixed thoroughly. Then, 2.6 vol of pre-chilled 96% Ethanol (Sigma) was added, mixed again, spun shortly and precipitated at À20C overnight. The next day the tubes were placed into a high-speed cent- rifuge pre-cooled to 4C, and centrifuged at 15 000 g for 1 h. The pellet was thoroughly washed by 70% EtOH (1 ml) at room temperature. During this procedure the pellet was detached from the bottom of the tube and it was centrifuged once more at 15 000 g for 40 min at room temperature. The supernatant (EtOH and salts) was aspirated using 1 ml filtered pipette tip. The tube was spun down, and the rest of the ethanol was aspirated completely using sequentially 30 and 10 ml filtered pipette tips without touching the pellet.
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