For PCR tests, DirectDetect™ SARS-CoV-2 Detection Kit (Coyote Bioscience Co, China) and Bio-Speedy SARS-COV2 (2019-nCoV) qPCR Detection Kit (Bioeksen R&D Technologies Ltd, Turkey) was used and according to the manufacturers’ recommendations. The PCR tests were performed with Qiagen Rotorgene Q-5 Plex-HRM Thermal Cycler (Qiagen, Belgium).
Rotor gene q 5 plex hrm thermal cycler
Manufactured by Qiagen
Sourced in Germany
The Rotor-Gene Q 5-Plex HRM thermal cycler is a real-time PCR instrument capable of performing high-resolution melt (HRM) analysis. It features a 5-channel detection system and can accommodate up to 72 samples in a rotor-disc format.
Automatically generated - may contain errors
6 protocols using rotor gene q 5 plex hrm thermal cycler
1
SARS-CoV-2 Detection Kit Protocols
2
Apoptotic Gene Expression Analysis
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Analyses of mRNA transcripts of key pro- and anti-apoptotic marker genes were conducted with MCF-7 cells exposed to 100, 400 or 800 µg/mL doses of F3 for 72 h [16 (link)]. Briefly, RNA was isolated by use of the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the instructions of the manufacturer. Assessments of RNA quality, genomic DNA elimination and cDNA synthesis were carried out as previously described [16 (link)]. Amplification programs and PCR amplicon specificity were performed and assessed by use of a Rotor-Gene Q 5-Plex HRM thermal cycler (Qiagen, Germany) with QuantiTect SYBR-Green PCR Kit (Qiagen, Germany) as previously documented following standard protocols [16 (link)]. The following primers were used: Hs_P53_1_SG QuantiTect Primer Assay (QT00060235); Hs_CASP3_1_SG QuantiTect Primer Assay (QT00023947); B-cell lymphoma 2 Hs_BCL2_1_SG QuantiTect Primer Assay (QT00025011); Bcl-2-like protein Hs_BAX_1_SG QuantiTect Primer Assay (QT00031192); and 18S rDNA house-keeping (HK) gene Hs_RRN18S_1_SG QuantiTect Primer Assay (QT00199367). PCR thermal cycling program and gene expression analysis to determine the fold-change relative to the 18S gene were essentially performed as previously reported [16 (link)].
3
Apoptosis Marker Gene Expression Analysis
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Analyses of gene expression of key pro-and anti-apoptotic marker genes were conducted with HepG-2, MCF-7, Bj-1 and MCF-12F cells exposed to IC50 obtained data (Table 3 ) for 72 h. Briefly, total RNA was extracted using the Total RNA Purification Kit (QIAGEN., Thorold, ON, Canada) according to manufacturer’s instructions. cDNA synthesis was carried out as previously described by Aboul-Soud et al. [61 (link)]. Amplification programs and PCR amplicon specificity were performed and assessed by the use of a Rotor-Gene Q 5-Plex HRM thermal cycler (Qiagen, Germany) with QuantiTect SYBR-Green PCR Kit (Qiagen, Germany) as previously documented following standard protocols [61 (link)]. The following primers were used: Hs_P53_1_SG QuantiTect Primer Assay (QT00060235); Hs_CASP3_1_SG Quan-tiTect Primer Assay (QT00023947); B-cell lymphoma 2 Hs_BCL2_1_SG QuantiTect Primer Assay (QT00025011); Bcl-2-like protein Hs_BAX_1_SG QuantiTect Primer Assay (QT00031192); and 18S rDNA house-keeping (HK) gene Hs_RRN18S_1_SGQuantiTect Primer Assay (QT00199367). PCR thermal cycling program and gene expression analysis to determine the fold-change relative to the 18S gene were essentially performed as previously reported [61 (link)].
4
Real-Time Quantitative PCR Analysis
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qRT‐PCR was performed using the Rotor-Gene Q 5plex HRM thermal cycler (Qiagen,
Germany). Each 15-μL reaction contained 100 ng cDNA, 0.3 μL gene-specific forward and
reverse primers (10 μM), and 7.5 μL Platinum Sybr Green qRT‐PCR SuperMix-UDG kit
(Invitrogen). The cycling conditions were as follows: initial template denaturation
for 5 min at 95°C, followed by 40 cycles of denaturation at 95°C for 20 s, annealing
at 60°C for 30 s, and extension at 72°C for 30 s. Fluorescence detection was
performed during each cycle at 72°C to identify the positive samples. Each sample was
assessed in triplicate, and controls without template were included in parallel for
each reaction. Amplification was followed by a melting curve analysis to check PCR
product specificity. The fold changes in gene expression relative to the levels
obtained in healthy rats, which were set equal to 1, were analyzed and calculated
using the 2-ΔΔct method (13 (link)).
Germany). Each 15-μL reaction contained 100 ng cDNA, 0.3 μL gene-specific forward and
reverse primers (10 μM), and 7.5 μL Platinum Sybr Green qRT‐PCR SuperMix-UDG kit
(Invitrogen). The cycling conditions were as follows: initial template denaturation
for 5 min at 95°C, followed by 40 cycles of denaturation at 95°C for 20 s, annealing
at 60°C for 30 s, and extension at 72°C for 30 s. Fluorescence detection was
performed during each cycle at 72°C to identify the positive samples. Each sample was
assessed in triplicate, and controls without template were included in parallel for
each reaction. Amplification was followed by a melting curve analysis to check PCR
product specificity. The fold changes in gene expression relative to the levels
obtained in healthy rats, which were set equal to 1, were analyzed and calculated
using the 2-ΔΔct method (13 (link)).
5
Multiplex real-time PCR for antibiotic resistance and virulence genes in S. aureus
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All S. aureus strains were analysed for detection of the mecA, mecC (methicillin resistance), ermA, ermC (erythromycin resistance), tetK, tetM (tetracycline resistance) and lukF-PV (virulence) genes by our designed multiplex real-time PCR protocols. The 16S rRNA coding sequence was applied as an internal PCR control. The primer and probe sequences were designed using the Vector NTI Advance™ program (Thermo Fisher Scientific, Inc., USA) for sequence alignment and FastPCR online (http://primerdigital.com/tools/pcr.html ) Java applet for primer tests (Table I ). The multiplex reactions were divided into the mixture I (targeting ermA, lukF-PV, ermC, and 16S rRNA), mixture II (targeting tetK, tetM, mecA, and 16S rRNA), and mixture III (targeting mecC and 16S rRNA). The reactions were carried out in a total volume of 15 μl with 1 μl of S. aureus lysate DNA. The multiplex real-time PCR mixture composition was 7.5 μl of 2 × SensiMix™ II Probe (Bioline Reagents Ltd., UK), 200 nM of each primer (Biolegio, The Netherlands), and 100 nM of each hydrolysis probe (Biolegio, The Netherlands). The reactions were performed using a Rotor-Gene Q 5plex HRM thermal cycler (QIAGEN, Germany) under the following conditions: initial denaturation at 95°C for 10 min (1 cycle) followed by 40 cycles of denaturation at 95°C for 20 s and primer annealing and extension at 55°C for 1 min.
6
Quantitative Analysis of LAPTM4B Expression
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The expression of LAPTM4B mRNA was quantified using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) technology on liver tissue samples of disease controls as well as tumor tissues and adjacent NTs obtained from patients in accordance with an earlier study. 15 LAPTM4B qRT-PCR was conducted on a Rotorgene Q, 5-Plex HRM Thermal Cycler (Qiagen GmbH, Hilden, Germany). Catalogues of total RNA extraction, cycling conditions for LAPTM4B qRT-PCR, and the interpretation of LAPTM4B mRNA expression have been discussed in Supplementary Materials and Methods.
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