Cfx96 pcr thermal cycler

Briefly, liquid-based cytology (LBC) samples were collected using broom-like devices with detachable heads (BD SurePathTM collection vial, Becton, Dickinson and Company, BD Life Sciences-Integrated Diagnostic Solutions, Sparks, MD, USA) according to the manufacturer’s instructions. The cervical broom was placed into the cervical canal and was rotated 360 degrees around the entire cervical canal. The detachable head was placed in a vial with preservative fluid. The LBC sample collection and transient storage temperature followed the manufacturer’s instructions.
Subsequently, the HPV test with Anyplex HPV28 assay using LBC specimens was performed according to the manufacturers’ instructions. DNA from each LBC sample was extracted using STARMag 96 X4 Universal Cartridge Kit (Seegene, Seoul, Korea). HPV genotyping was conducted using a multiplex polymerase chain reaction (PCR) using a CFX96 PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA), according to manufacturers’ guidelines, using 5 μL of template DNA in a total volume of 20 μL for Anyplex™ II HPV28.
At the initial examination and at least once during a follow-up visit, HPV testing with genotyping and semi-quantitative VL measurement were conducted according to the manufacturer’s instructions using the Anyplex^TM II H28 Detection kit (Cat No. HP7S00X, Seegene, Seoul, Korea).

The Lim broth was purchased from Becton Dickinson (Franklin Lakes, NJ, USA). The Presto™ Mini gDNA Bacteria Kit was purchased from Geneaid Biotech Ltd. (New Taipei City, Taiwan). The QIAamp DNA Micro Kit was purchased from QIAGEN (Hilden, Germany). Oligonucleotides were synthesized by Integrated DNA Technologies Biotech Co., Ltd. (Coralville, IA, USA). The oligonucleotide sequences utilized in this study are detailed in Table 1. The Bst 2.0 WarmStart and buffer were purchased from New England Biolabs (Ipswich, MA, USA). The nucleic acid test lateral flow strip was acquired from Panion & BF Biotech Inc. (New Taipei City, Taiwan). The other components were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were utilized as received and without additional purification. Melting curve analysis and LAMP were carried out on the CFX96 PCR Thermal Cycler from Bio-Rad (Hercules, CA, USA). Genomic DNA quantification was performed using EzDrop-1000 from Blue-Ray Biotech (New Taipei City, Taiwan).

Total RNA was extracted using the Trizol reagent and reversed transcribed. The RNA quality was confirmed by agarose gel electrophoresis and the Agilent 4,150 Tapestation system. Real–time PCR was performed in 96-well plates using the Luna universal PCR mix in a CFX96 PCR thermal cycler (Biorad). The thermal cycling conditions comprised an initial denaturation step at 95°C for 60 s followed by 42 cycles at 95°C for 15 s and 60°C for 30 s. The comparative Ct method was used to calculate the relative gene expression (37 (link)). Expression levels of the genes of interest were normalized to the respective levels of GAPDH. Supplementary Table 1 shows the sets of real-time qPCR (RT-qPCR) primers used for each gene whose expression was studied.