Measurements of Human Papillomavirus Transcripts by Real Time Quantitative Reverse Transcription-Polymerase Chain Reaction in Samples Collected for Cervical.

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Measurements of Human Papillomavirus Transcripts by Real Time Quantitative Reverse Transcription-Polymerase Chain Reaction in Samples Collected for Cervical Cancer Screening Laurence Lamarcq, James Deeds, David Ginzinger, Jean Perry, Siddhartha Padmanabha, Karen Smith-McCune The Journal of Molecular Diagnostics Volume 4, Issue 2, Pages 97-102 (May 2002) DOI: 10.1016/S1525-1578(10)60687-3 Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 RNA from cells scraped from the cervix demonstrates variable degrees of degradation regardless of collection method. Cells scraped from the cervix were collected directly into Trizol (lanes 1 and 2), into DME (lanes 3 and 4) or into 95% ethanol (lanes 5 and 6) and immediately snap-frozen in liquid nitrogen. RNA was extracted with Trizol and analyzed by Agilent biosizing gel electrophoresis. RNA quantity was calculated from the absorbance at 260 nm, and 1 μl (approx 100 ng) was loaded per lane. Ribosomal RNA was used as the molecular weight maker (lane 0); positions of 28S and 18S rRNA are indicated on the left. Arrow(right) indicates 200 bp. The Journal of Molecular Diagnostics 2002 4, 97-102DOI: (10.1016/S1525-1578(10)60687-3) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Direct comparisons of RNA yield, purity, and integrity using different RNA extraction procedures. Pairs of clinical samples were pooled, divided into two equal aliquots, and extracted in parallel with RNeasy or Trizol. RNA concentration and yield were calculated from absorbance at 260 nm. In A, values in italics were obtained after an additional acidic phenol-chloroform extraction of the Trizol-extracted RNA. RNA size was assessed by Agilent gel electrophoresis of samples extracted with RNeasy (B), and Trizol (C). One ul was added to each lane, corresponding to approximately 25 ng (B) and 100 ng (C). Numbers 1 to 3 above each lane in B and Ccorrespond to the samples number in A; matched numbers represent identical aliquots extracted in parallel. Ribosomal RNA was used as the molecular weight maker (lane 0); positions of 28S and 18S rRNA are indicated on the left. Arrow(right) indicates 200 bp. The Journal of Molecular Diagnostics 2002 4, 97-102DOI: (10.1016/S1525-1578(10)60687-3) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Efficient and linear measurements of housekeeping genes in clinical samples collected for cervical cancer screening by TaqMan.A: Increasing amounts of RNA in the RT reaction were used to measure GAPDH amplification using RNA extracted by RNeasy or Trizol/phenol-chloroform. The “no RT” controls contained the highest amount of RNA that was used in the RT reaction, but the RT enzyme was omitted. Each data point indicates average of duplicate determinations with error bars corresponding to the SD. Numbers next to the lines indicate the slopes. B: Simultaneous amplification of GAPDH (circles) and GUS (triangles) using RNA extracted with Trizol/phenol-chloroform from a single clinical sample. The “no RT” control contained the highest amount of RNA that was used in the RT reaction, but the RT enzyme was omitted. Each data point indicates average of duplicate determinations with error bars corresponding to the SD. Numbers next to the lines indicate the slopes. The Journal of Molecular Diagnostics 2002 4, 97-102DOI: (10.1016/S1525-1578(10)60687-3) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Measurement of HPV 16 and 18 E7 in cervical cancer cell lines by TaqMan. The specificity of E7 primers for their respective HPV types were tested using RNA extracted from both Caski (HPV 16) and HeLa (HPV 18) cells (white bars). “No RT” controls (dark gray bars) were corresponding reactions in which the RT enzyme was omitted. Each data point indicates average of duplicate determinations with error bars corresponding to the SD. The Journal of Molecular Diagnostics 2002 4, 97-102DOI: (10.1016/S1525-1578(10)60687-3) Copyright © 2002 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions