DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

Abstract

The noncritical use of housekeeping genes, RNA mass, or cell number for normalization in quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays has come under scrutiny in recent years, highlighting the need to evaluate references in the immediate context of the relevant samples and experimental design. The purpose of this study was to select appropriate references for normalizing qRT-PCR assays of gene expression in exfoliated cervical cells. We used total nucleic acid extracts from 30 samples, representing the full spectrum of pre-invasive cervical neoplasia. We determined the DNA content by quantitative PCR for the single-copy gene ␤-globin and total RNA content using quantitative image analysis of ribosomal bands. In addition, qRT-PCR for 13 candidate housekeeping genes was performed. We used two analysis methods, geNorm and NormFinder, to identify the best combination of reference genes and then correlated housekeeping gene expression with DNA content and gel representation of ribosomal RNA. ACTB was the most stable single gene. The addition of PGK1 and RPLP0 increased the robustness in qRT-PCR applications not stratified by disease. These genes also showed the highest correlation with DNA contents in the same samples. If special attention to intraepithelial lesions is appropriate , RPL4 and PGK1 are recommended as the best combination of two genes.

Evaluation of RNA Markers for Early Detection of Cervical Neoplasia in Exfoliated Cervical Cells

“…We verified target specificity of each primer pair by gel electrophoresis of amplicons generated from cDNA from pooled cervical exfoliated cells, human genomic DNA (Roche Diagnostics, Indianapolis, IN), and a-no template control. Primers and conditions for the reference gene transcripts PGK1 and RPL4 as well as the external control CAB were established previously (16). After amplification, all PCR reactions were subjected to dissociation curve analysis to verify product specificity by a single peak of the second derivative of the melting curve.…”

“…was previously determined to be the most stable combination of transcripts in exfoliated cervical cells (16).…”

p16INK4a/Ki‐67 co‐expression specifically identifies transformed cells in the head and neck region

“…Quantitative realtime polymerase chain reaction was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, United Kingdom) and HPV16 E6*I forward/reverse primer mix. 32 Expression of the house-keeping gene actin 33 was analyzed for reference purposes. All samples were run in triplicates for both E6*I and actin cDNA in the quantitative real-time PCR.…”

Epstein-Barr Virus Transcytosis through Polarized Oral Epithelial Cells

“…The target gene was amplified from cDNA using SYBR green real-time PCR master mix (Life Technologies, Grand Island, NY), a 1 mM concentration of each primer, and 5 l of the cDNA in a total volume of 20 l. The following primers were used: LMP1 (forward, 5=-CAG TCA GGC AAG CCT ATG A-3=; reverse, 5=-CTG GTT CCG GTG GAG ATG A-3=), EBNA1 (forward, 5=-TAC AGG ACC TGG AAA TGG CC-3=; reverse, 5=-TCT TTG AGG TCC ACT GCC G-3=, gp350/220 (forward, 5=-AGA ATC TGG GCT GGG ACG TT-3=; reverse, 5=-ACA TGG AGC CCG GAC AAGT-3=), and BZLF1 (forward, 5=-AAA TTT AAG AGA TCC TCG TGT AAA ACA TC-3=; reverse, 5=-CGC CTC CTG TTG AAG CAG AT-3=). The beta-2-microglobulin gene was used as an internal control (forward, 5=-TGA CTT TGT CAC AGC CCA AGA TA-3=; reverse, 5=-CGG CAT CTT CAA ACC TCC A-3=) (70). EBV gene expression was calculated by the ⌬⌬CT method (71,72).…”