The regulatory region of the human ubiquitin C promoter (1204 bp) was amplified by PCR, the vector pFUGW (Addgene) was used as the template as well as specific primers (Invitrogen). The fragment was then inserted into the promoterless vector pDsRed2-1 (Clontech). PCR of the YFP-I152L gene (720 bp) was performed using vector pcDNA3.1-YFPI152L (Invitrogen) as a template and specific primers (Invitrogen). The YFP-I152L-coding construct was kindly provided from Prof. Joe Lynch (Queensland Brain Institute, The University of Queensland, Brisbane, Australia). After removing the sequence of the red fluorescent protein from the vector phuUbC-DsRed2, YFP-I152L was sub-cloned into the vector. The expression of YFP-I152L is under the control of the constitutive active human ubiquitin C promoter, the plasmid carries the neomycin resistance gene for the selection with the antibiotic geneticin.
Specific primers
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Sourced in United States, China, Germany, Japan
Specific primers are laboratory reagents used in molecular biology techniques such as Polymerase Chain Reaction (PCR) to selectively amplify target DNA sequences. They are short, single-stranded DNA molecules designed to bind to and initiate the replication of specific genetic targets.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Gotaq 2 step rt qpcr system, by Promega (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Thermo Fisher Scientific (3 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Kod fx neo, by Toyobo (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rna extraction kit, by Qiagen (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Perfect super green super mix, by Quanta Biosciences (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
81 protocols using specific primers
1
Ubiquitin C Promoter-Driven YFP-I152L Expression
2
Quantitative Analysis of COL4A5 Transcripts
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Total RNA was extracted from cells using an RNA Isolation Kit (Zymo Research, Irvine, CA). RNA integrity was assessed after agarose gel electrophoresis, and the concentrations were measured spectrophotometrically (Nanodrop Technologies, Wilmington, DE). The samples were subjected to DNase treatment using a DNA-free kit (Ambion, Inc., Austin, TX), and 1 μg from each sample reverse-transcribed using oligo dT and a SuperScript III First Strand Synthesis System Kit (Invitrogen).
Samples were then assayed for COL4A5 transcripts, using the fluorescent intercalating agent SYBR Green 1 (Qiagen, Hilden, Germany), specific primers (Life Technologies, Mulgrave, Victoria, Australia) (Supplementary Tables S1 and S2 ), and the ABI 7500 real-time PCR System (Applied Biosystems, Waltham, MA). Individual reactions comprised 5 μl of 2× QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.7 μl each of 20 ng/μl sense and antisense primer, and 2 μl of 100 ng/μl cDNA template, in a total volume of 10 μl. The threshold cycle value was calculated at the end of each run using glyceraldehyde-3-phosphate dehydrogenase as the internal control and software provided by the manufacturer. Each sample was examined in duplicate and the assays performed in triplicate. The results were compared with expression in the 3 normal male fibroblast cell lines in different experiments.
Samples were then assayed for COL4A5 transcripts, using the fluorescent intercalating agent SYBR Green 1 (Qiagen, Hilden, Germany), specific primers (Life Technologies, Mulgrave, Victoria, Australia) (
3
Quantitative PCR Analysis of Monocyte Subsets
4
Quantitative RT-PCR Analysis of Rat Retinal RNA
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Total RNA from rat retinas were extracted using RNeasy Plus Mini kit from Qiagen (Courtaboeuf, France). To ensure the absence of DNA, a treatment with RNase-free DNase (Qiagen) and a subsequent cleanup with the RNeasy MinElute cleanup kit (Qiagen) were performed. This was confirmed by subjecting 10 ng of each RNA preparation to qPCR with human ND4, endogenous ATP6 or BRN3A primers. One microgram of total RNA was reverse transcribed with oligo-dT using Superscript II Reverse Transcriptase (Life Technologies, Saint-Aubin, France). Quantitative PCR reactions were performed using ABI 7500 Fast (Applied Biosystems, Saint Aubin, France) and the specific primers (Life Technologies) listed in Supplementary Table S3 . The equivalent of 10 and 2 ng of cDNAs were used per gene as template for qPCR reactions with Power Sybr green PCR Master Mix (Applied Biosystems). Each biological sample was subjected to the assay in triplicates per gene; Ct values (number of cycles required for the fluorescent signal to cross the background threshold) were obtained with the ABI 7500 software (v.2.0.4). To determine the relative mRNA amount of each studied gene we used the comparative ΔΔCt method and the mitochondrial ATP6 gene as normalizing gene since its mRNA steady-state levels remained almost unchanged in all the samples evaluated.
5
Isolation and Quantification of RNA
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The total cellular RNA was isolated from L6-GLUT4 cells and gastrocnemius muscle and the brain of wild-type C57BL/6 mice using the TRIzol® RNA isolation reagent (Life Technologies (CA, USA)) according to the manufacturer’s instructions. cDNAs were synthesized using the SuperScript III first-strand synthesis system for RT-PCR (Life Technologies) and then amplified using KOD FX neo (Toyobo (Japan)) and specific primers (Life Technologies) (5’-GGCAGTTGGTACGACAGGAT-3’ and 5’-CGAAGCGCAGTTTACTTTCC-3’ for FLJ00068, and 5’-CCCCTTCATTGACCTCAACTAC-3’ and 5’-ATGACCTTGCCCACAGCCTTGG-3’ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) according to the manufacturer’s instructions. Genome sequences recognized by the above primers are identical between rat and mouse.
6
Transcriptional regulation in mouse hippocampus
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Total RNA extraction, reverse transcription and real-time quantitative PCR (RT-qPCR) amplification assays for PSD95, REST and PTBP1 were performed using specific primers (Life Technologies) as previously reported (Tajeddinn et al. 2016 (link)). mRNA copy numbers of all target transcripts were adjusted by mRNA copy numbers of GAPDH and the values calculated were compared with controls (set at 100%). Expression of miR-124a and miR-9 was measured in the whole hippocampus of Cyp27Tg and WT mice using stem-loop specific primers for mmu-miR-124a or mmu-miR-9 (Life Technologies) as previously reported (Jimenez-Mateos et al. 2015 (link)). Expression of RNU19 was used for normalization. A relative fold change in expression of miR-124a and miR-9 was determined using the 2−∆∆CT method.
7
Quantitative PCR Analysis of Gene Expression
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Total RNA extraction was performed using the TRIzol reagent (Life Technologies, Rockville, MD), according to the manufacturer's instructions. Amplification of transcripts was performed using 3–5μg/μl of total RNA. The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using moloney murine leukemia virus reverse transcriptase (MMLV) and oligo-d(T)15 primer. Specific primers were purchased from Life technologies and listed in S1 Table . The cDNA samples were 2.5 X diluted by H2O as template for quantitative PCR assay. QPCR reactions were carried out in triplicate on 96-well plate using an Applied Biosystems 7500Fast Real-Time PCR System. To calculate the relative transcriptional expression, the Ct values of interested genes were normalized by average Ct values of gapdh as ΔCt, then the ΔCt of MycNkd cells were normalized by ΔCt of control cells to get the ΔΔCt. The relative transcriptional expression of interested genes was indicated with2(-ΔΔCt).
8
Quantitative Analysis of Oral Bacterial Species
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The amount of each bacterial species and of eubacteria was evaluated by qPCR using specific primers (Life Technologies) and TaqMan probes (Life Technologies or Roche; Table S1). First, DNA was purified with the QIAamp DNA Mini Kit (Qiagen GmbH) following the manufacturer’s instructions for buccal swabs. Then, reactions were performed as previously described [20 (link)], and the resulting crossing points were transformed to number of copies/µl through plasmid standard curves. Each standard plasmid was prepared by cloning the target PCR products in pGEMT [21 ] with the specific primers from Table S1, using DNA from E. brachy DSM 3990, F. alocis ATCC 35,896, F. fastidiosum DSM 25,557, P. endodontalis ATCC 35,406, P. gingivalis ATCC 33,277, S. sputigena ATCC 35,185, T. forsythia ATCC 43,037, T. denticola DSM 14,222 and T. socranskii ATCC 35,534 as templates. The number of copies/µl was estimated using a Nanodrop ND-1000 UV – vis spectrophotometer (Nanodrop Technologies).
Eubacterial load was used to normalise the data between individuals. The standard curve was prepared as in Àlvarez et al. [20 (link)], using DNA from Streptococcus gordonii ATCC 49,818, Veillonella parvula NCTC 11,810, Actinomyces naeslundii DSM 17,233, Fusobacterium nucleatum DSM 20,482 and P. gingivalis ATCC 33,277 as templates.
Eubacterial load was used to normalise the data between individuals. The standard curve was prepared as in Àlvarez et al. [20 (link)], using DNA from Streptococcus gordonii ATCC 49,818, Veillonella parvula NCTC 11,810, Actinomyces naeslundii DSM 17,233, Fusobacterium nucleatum DSM 20,482 and P. gingivalis ATCC 33,277 as templates.
9
Quantifying Lgr5 and SOD1 Gene Expression
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Total RNA from tumor cells was isolated by an RNA extraction kit (Qiagen) and cDNA synthesis and real-time (RT)-PCR were performed using the first strand cDNA synthesis kit (Fermentas) and SYBR Green Master Mix kit (Roche) applying specific primers (Life Technologies) for human leucine-rich-repeat-containing G-protein-coupled-receptor 5 (Lgr5) and superoxide dismutase 1 (SOD1). Transcript levels of the gene of interest were normalized to the expression of glyceraldehy-3-phosphat-dehydrogenase (GAPDH). Primer sequences are listed in S2 Table .
10
Gene Expression Profiling by qRT-PCR
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cDNAs were generated using 10 ng of RNA and specific primers (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. qRT-PCR reactions were performed using TaqMan chemistry and the ABI 96-well platform (Life Technologies). Transcripts were tested, analyzed and normalized relative to housekeeping miRNAs (RNU24, and U6 using ExpressionSuite® v1.0.3 software, Life Technologies).
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