Cells were collected, lysed and total RNA was extracted using the RNeasy Mini kit (Qiagen, Germany); RNA was quantified using MySpec (VWR, PA, USA), and cDNA synthesis was carried out using the High-capacity RNA-to-cDNA kit on a thermal cycler (VWR, PA, USA). TaqMan probes of interest were incubated with cDNA samples in Fast Advanced Master Mix. For all samples, GAPDH TaqMan probes were included as housekeeping gene reference. Samples were run on a BioRad CFX96 Touch Real-Time PCR Detection System using the multiplex option for superior Ct quantification. Depending on the graph presented, either ΔCt or ΔΔCts were determined. Samples that lacked Ct values (e.g., RELN, GFAP for all NES lines) or had values over 35 were assigned a Ct value of 35 for the ΔΔCt analysis to be possible (or to minimize overestimation of results). PCA was done with R using the prcomp function (R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/ ) and hierarchical clustering with the Morpheus software using average linkage clustering, person correlation (Morpheus, https://software.broadinstitute.org/morpheus ). A list of the TaqMan probes used can be found in Supplementary Table 4.
Thermal cycler
Manufactured by Avantor
Sourced in Germany, United States, United Kingdom
A thermal cycler is a laboratory instrument used for the amplification of DNA sequences through the Polymerase Chain Reaction (PCR) process. It precisely controls the temperature of the samples to facilitate the repeated thermal cycling required for DNA replication.
Automatically generated - may contain errors
Lab products found in correlation
Peqlab total rna isolation kit, by Avantor (8 mentions)
Lightcycler 480, by Roche (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (4 mentions)
Quantitect primer assay hs gapdh, by Qiagen (4 mentions)
Gene specific primers, by Thermo Fisher Scientific (4 mentions)
Gotag qpcr master mix, by Promega (4 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Primers for real time pcr, by Thermo Fisher Scientific (3 mentions)
Rest software, by Qiagen (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnx plus kit, by CinnaGen (2 mentions)
Taq dna polymerase, by Merck Group (2 mentions)
Dual gel vertical electrophoresis system, by Avantor (2 mentions)
Myspec, by Avantor (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
E z n a tissue dna kit, by Omega Bio-Tek (1 mentions)
Twincubator, by Hain Lifescience (1 mentions)
Taq dna polymerase, by CinnaGen (1 mentions)
21 protocols using thermal cycler
1
Quantitative Transcriptomics via RT-qPCR
2
DNA Extraction and COI Gene Amplification
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A DNA extraction kit (E.Z.N.A. Tissue DNA Kit, Omega Bio-tek) was used for extracting DNA, following the manufacturer’s protocol. Success of DNA extraction was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific). The universal primers HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’ ) and LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’ ), described by Folmer et al. [37 (link)], were used for the amplification of the COI gene fragment. PCR amplification was performed in a 25 μL reaction mixture containing 2 μL template DNA, 15.925 μL DNA free water, 1 μL (0.4 μM) of each forward and reverse primer, 2.5 μL (1 mM) PCR buffer, 0.5 μL (0.2 mM) dNTP, 0.875 μL (3.5 mM) MgCl2, 1 μl (10 mg/ml) BSA and 0.2 μL of (5 U/μL) Taq DNA polymerase, using a UNO96 VWR Thermal cycler. The following temperature profile was used: 5 min at 94 °C, followed by 40 cycles of 30 sec at 94 °C, 1 min at 51 °C and 1 min at 72 °C. Final extension was conducted at 72 °C for 10 min. After checking if the amplification was successful with gel electrophoresis on a 2% TBE agarose gel, fragment sequencing was carried out using an ABI 3730XL sequencer.
3
Molecular Detection of TB Drug Resistance
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The identification of genotypic drug resistance to RIF (based on mutations the rpoB gene) and INH (based on mutations in katG and inhA genes) was carried out using the manufacturer’s instruction27 in the TB Laboratory at ALIPB-AAU. Accordingly, 50 µL of PCR mixture consisting of 10 µL AM-A GT MTBDRplus ver 2.0, 35 µL AM-B GT MTBDRplus ver 2.0 and 5µL DNA template from an isolate was used to perform this assay. M. tuberculosis H37Rv DNA template was used as a positive control. Water (Qiagen NV, Venlo, the Netherlands, product) served as a negative control. A thermal cycler (VWR, Leicestershire, UK) was programmed as follows: 15 min for enzyme activation at 95°C followed by 10 cycles of 30 sec denaturation at 95°C, 2 min annealing at 58°C, 20 cycles of 25 sec denaturation at 95°C, 40 sec annealing at 53°C, 40 sec elongation at 70°C, and finally elongation at 70°C for 8 min. Biotin-labeled amplicons were hybridized to DNA probes attached to a DNA strip®. Hybridization was done using the TwinCubator (Hain Lifescience, Nehren, Germany). Results were interpreted based on the presence and absence of wild-type (WT) and mutation (MUT) probes.
4
Genomic DNA Extraction and pslA Gene Amplification
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Genomic DNA was extracted by boiling (19 (link)). PCR amplification of the pslA gene was carried out using the following primers: pslA-F, 5’-CACTGGACGTCTACTCCGACGATAT-3’; pslA-R, 5’-GTTTCTTGATCTTGTGCAGGGTGTC-3’ (Bioneer, Korea), generating an amplification product of 1119 bp (20 (link)). The reaction mixture (25 µL) contained 1 µL of the extracted DNA, 1.5 mM MgCl2, 0.4 mM of each dNTP, 10 pM of each primer and one unit of Taq DNA polymerase (CinnaGen, Iran). Amplifications were performed in a thermal cycler (Peqlab, Germany) using the following program: an initial incubation at 94°C for 10 minutes, followed by 30 cycles of one minute denaturation at 94°C, 30 seconds annealing at 55°C and one minute extension at 72°C followed by 10 minutes at 72°C. The amplification products were separated on 1% agarose gels, stained with Red Safe (Intronbio, Korea), and visualized using an image analysis system (UVItec, St John’s Innovation Centre, UK).
5
Pluripotency Gene Expression Analysis in hAFSCs
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Total RNA was extracted from 5–6×105 undifferentiated hAFSCs from 3th-5th passages, using RNX-Plus kit (CinnaGen, cat# RN7713C) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from total RNA using Termo Scientific Revert Aid First Strand cDNA Synthesis Kit (Fermentas, cat# K1621) with oligo-dT primer in a 20 μl reaction mixture which was performed in a Thermal Cycler (PeQLab). Primers used for amplification are listed in Table 1 . All samples were checked for b-actin (housekeeping gene) as an internal control to verify the achievement of the RT reaction and then other pluripotency genes were checked with its specific primers. RT-PCR using the Hyper Script RT master mix (GenAll, cat# 601–710) was performed with 2μl of the single stranded cDNA sample for detection of NANOG and Oct4 (POU5F1) gene expression. After initial denaturation at 94°C for 4 min, PCR amplification was carried out at 94°C for 30 sec, followed by annealing temperature at 56°C for 30 sec, and 72°C for 30 sec and extension at 72°C for 5 min for a total of 35 cycles. Amplified PCR products were separated on a 2% agarose gel by electrophoresis and the bands were visualized by dying buffer and photographed with a gel doc.
6
Quantitative Analysis of BAG1 Expression
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The cDNA was synthesized from total RNA using Prime Script RT-PCR Kit (Takara, Kusatsu, Japan), according to the manufacturer’s instruction. Briefly, 4 µL of 5× buffer, 1 µL of oligo(dT) primer, 1 µL of RT enzyme mix, 1 µL of random hexamers, 10 µL of the total extracted RNA, and 3 µL of sterile DW were mixed in a sterile microtube to reach a total volume of 20 µL. The mixture was incubated in thermal cycler (Peqlab, Fareham, UK) for one cycle at 37°C for 15 minutes, one cycle at 85°C for 5 seconds, and one cycle at 4°C for 5 minutes. Furthermore, random samples without RT enzymes (no RT controls) were used to detect gDNA contamination in the samples. Primers of BAG1 (bradyzoite surface antigen 1) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) have been used for real-time PCR as reported in a previous study by the authors.36 (link) Reaction conditions included a cycle at 50°C for 2 minutes, a cycle at 95°C for 15 minutes followed by 40 cycles at 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. A nontem-plate control was used as negative control, and all reactions were carried out in triplicate to ensure reproducibility. The relative gene expression level for BAG1 in each sample was normalized by subtracting the cycle threshold (CT) of the housekeeping gene (GAPDH) to calculate the ΔCT.37 (link)
7
MICU1 Knockdown and Overexpression Protocol
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Total RNA was isolated according to manufacturer’s protocol using the PEQLAB total RNA isolation kit (Peqlab, Erlangen, Germany). cDNA was generated using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and a thermal cycler (Peqlab). Knockdown and overexpression efficiency of MICU1 was examined by RT-PCR using a QuantiFast SYBR Green RT-PCR kit (Qiagen, Hilden, Germany) and a LightCycler 480 (Roche Diagnostics, Vienna, Austria). Relative expression of the gene of interest was normalized to GAPDH, as a reference gene. Primers for real-time PCR were designed by us and obtained from Invitrogen (Vienna, Austria). The respective primer sequences used can be found elsewhere (Supplementary Table. 1 ).
8
Quantifying Gene Expression in HeLa Cells
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Total RNA was isolated using the PEQLAB total RNA isolation kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany), and reverse transcription was performed in a thermal cycler (PEQLAB Biotechnologie GmbH) using a cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Expression of MCU, UCP2, UCP3, Letm1, and AKAP-RFP-CAAX in HeLa cells was examined by RT-PCR. A QuantiFast SYBR Green RT-PCR kit (Qiagen, Hilden, Germany) was used to perform real time PCR on a LightCycler 480 (Roche Diagnostics, Vienna, Austria), and data were analyzed by the REST Software (Qiagen). Relative expression of specific genes was normalized with GAPDH as a housekeeping gene. Primers for real time PCR were obtained from Invitrogen (Vienna, Austria), and their sequences (5′-3′) were:
9
Genotypic Identification of Probiotic Bacteria
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The bacterial isolates that fulfilled the selection criteria for probiotics were finally chosen as probiotic candidates to be identified genotypically using the 16S rRNA gene amplification method.
The PCR reaction mixture with a total volume of 25 microliters, consisted of 10 pmol primers, and the PCR master mix reaction mixture. The following universal primers; 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1492R (5′ GGT TAC CTT GTT ACG ACT T 3′) were used in the reaction.
PCR program in the thermal cycler (peQlab, United States) was comprised of initial denaturation at 95°C for 10 min followed by 35 cycles containing the second denaturation at 95°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min and 30 s, followed by a final extension step at 72°C for 10 min.
PCR products were detected and visualized by agarose gel electrophoresis (1% w/v) and subsequently sequenced. The resulting Sanger sequencing data were employed by the basic local alignment search tool (BLAST) to obtain sequence similarities. Thereafter, the sequences were registered in the NCBI1 database and assigned with accession numbers.
The PCR reaction mixture with a total volume of 25 microliters, consisted of 10 pmol primers, and the PCR master mix reaction mixture. The following universal primers; 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1492R (5′ GGT TAC CTT GTT ACG ACT T 3′) were used in the reaction.
PCR program in the thermal cycler (peQlab, United States) was comprised of initial denaturation at 95°C for 10 min followed by 35 cycles containing the second denaturation at 95°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min and 30 s, followed by a final extension step at 72°C for 10 min.
PCR products were detected and visualized by agarose gel electrophoresis (1% w/v) and subsequently sequenced. The resulting Sanger sequencing data were employed by the basic local alignment search tool (BLAST) to obtain sequence similarities. Thereafter, the sequences were registered in the NCBI
10
Gene Expression Analysis in NRK Cells
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NRK cells were transfected with IP3R-3 siRNA (see below) using Transfast (Promega Corp) using manufacturer's instructions. Total RNA was isolated using the PEQLAB total RNA isolation kit (Peqlab), and reverse transcription was performed in a thermal cycler (Peqlab) using a cDNA synthesis kit (Applied Biosystems). mRNA levels were examined by qRT-PCR. A QuantiFast SYBR Green RT-PCR kit (Qiagen) was used to perform real time PCR on a LightCycler 480 (Roche Diagnostics), and data were analyzed by the REST Software (Qiagen; https://www.qiagen.com ). Relative expression of specific genes was normalized to human GAPDH as a housekeeping gene. Primers for real time PCR were obtained from Invitrogen.
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