RNA was reverse-transcribed to cDNA using the Superscript VILO cDNA reverse transcription kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. TaqMan PCR primers were obtained as proprietary pre-optimized reagents (Life Technologies, Carlsbad, CA, USA), and probes were obtained from the Universal Probe Library (Roche, Basel, Switzerland). PCR primers and probes were combined with TaqMan gene expression master mix in real-time PCR reactions (7900HT Fast Real-Time PCR System; Applied Biosystems, Waltham, MA). The relative quantitation of mRNA expression was performed using the 2∆∆Ct method relative to the housekeeping gene (18S rRNA).
Taqman pcr primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan PCR primers are synthetic DNA oligonucleotides designed for use in polymerase chain reaction (PCR) assays. They function as specific probes that bind to target DNA sequences and enable the detection and quantification of those sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Dual luciferase assay reagent, by Promega (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Enzalutamide, by MedKoo Biosciences (1 mentions)
3h mibolerone, by PerkinElmer (1 mentions)
Mg132, by R&D Systems (1 mentions)
Ar n 20, by Santa Cruz Biotechnology (1 mentions)
R1881, by PerkinElmer (1 mentions)
Ar c 19, by Santa Cruz Biotechnology (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
M mlv rt, by Promega (1 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (1 mentions)
Taqman genotyper software, by Thermo Fisher Scientific (1 mentions)
12 protocols using taqman pcr primers
1
Quantitative RT-PCR analysis of mRNA
2
Androgen Receptor Signaling Assay
3
TaqMan RT-PCR for miRNA expression
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All reagents including specific and control primers for TaqMan real-time RT-PCR were purchased from Applied Biosystems and the reactions were set according to the manufacturer’s protocol. Briefly, total RNA was purified by TRIzol (Life Technologies/Invitrogen). 10 ng of total RNA and RT primers for hsa-miR-132, hsa-miR-212 and the housekeeping gene RNU44 were used for reverse transcription (RT) with TaqMan MicroRNA Reverse Transcription Kit. Real-time PCR quantification was performed using TaqMan PCR primers and TaqMan Universal PCR Master Mix, No AmpErase UNG on the ABT PRISM 7900HT Fast PCR system (Applied Biosystems). Relative mRNA expression was determined as previously described [26] (link).
4
Multiplexed miRNA and mRNA Expression Profiling
5
Affymetrix Gene Expression Profiling of K11bL Cells
6
Genotyping of Genetic Variants
7
Quantitative Analysis of p16Ink4a Expression
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Total cellular RNA was extracted from about 20,000 sorted HSCs using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA yield and quality were determined by measuring absorbencies at 260 and 280 nm, respectively. First-strand cDNA was synthesized from total RNA by RevertAid First Stand cDNA Synthesis Kit (Thermo scientific) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using TaqMan PCR primers and MasterMix (Applied Biosystems). Taqman MGB probes for the p16Ink4a (catalog number: Mm00494449_m1), and- the housekeeping gene Gapdh (catalog number: Mm99999915_g1) were obtained from Applied Biosystems (Foster City, CA). All samples were analyzed in triplicate using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). The threshold cycle (CT) values for each reaction were determined and averaged using TaqMan SDS analysis software (Applied Biosystems). The changes in gene expression were calculated by the comparative CT method (fold changes = 2−△△CT) as described previously [41 (link)].
8
Quantitative Analysis of miRNA and mRNA Expression in Multiple Myeloma Cells
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Total RNA from MM cell adhesions was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA). To analyze miRNA expression, we used the miRNA‐specific Taqman MicroRNA Assay Kits (Applied Biosystems, Foster City, CA). miRNA‐enriched RNA was first reverse‐transcribed with the Taqman microRNA Reverse Transcription Kit (Applied Biosystems), and quantitative polymerase chain reaction (PCR) was performed in triplicate with corresponding Taqman PCR primers (Applied Biosystems), and Taqman Universal Master Mix, according to manufacturer's instructions. The RNU44 small RNA was used for normalization. To analyze mRNA expression in MM cells by quantitative PCR we followed the described method [16 (link)], and the oligonucleotide sequences are listed in Table S1 . Control and α4 siRNAs were obtained from Sigma‐Aldrich and nucleofected as we previously described [16 (link)]. After 24 h, extent of α4 silencing was tested by flow cytometry. miRIDIAN miRNA Hairpin inhibitor for miR‐324‐5p and a Hairpin inhibitor negative control (40 nmol/L) (Dharmacon) were transfected by nucleofection (Amaxa, Cologne, Germany) into NCI‐H929 cells, using kit V and program T‐01.
9
Quantifying Gene Expression Levels
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The gene expression levels of matrix metalloproteinase 2 (MMP-2), MMP 9, hypoxia-inducible factor (HIF-1α), and vascular endothelial growth factor–A (VEGF-A) were measured using quantitative reverse transcription PCR (qRT-PCR). Total RNA was isolated using TRI Reagent Solution (Applied Biosystems, Carlsbad, CA, USA/Ambion, Austin, TX, USA) on days 2, 4, and 6. Next, 1 mg of total RNA was reverse-transcribed to complementary DNA (cDNA) using a Reverse Transcription System (Promega, Madison, WI, USA). Finally, an ABI 7300 Sequence Detection System (Applied Biosystems) was used to conduct qRT-PCR using TaqMan Universal PCR Master Mix and gene-specific TaqMan PCR primers (Applied Biosystems) (Table 1 ): MMP-2 (NM_001127891.1), MMP-9 (NM_004994.2), HIF-1α (NM_001530.3), and VEGF-A (NM_001025366.2). Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative threshold cycle (ΔΔCT) method of quantification.40 (link) All experiments were performed with four technical replicates.
10
Transcriptional Profiling of Hematopoietic Stem Cells
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Bone marrow Lin−Sca1+cKit+ (LSK) cells were sorted and collected in buffer RLT plus (Qiagen). These samples were then processed for RNA isolation and cDNA preparation at the University of Rochester Functional Genomics Core. Total RNA was isolated from sorted LSK cells using an RNeasy Mini Kit (Qiagen). RNA was preamplified and cDNA was produced using a WT-Ovation PicoSL kit (Nugen). For analysis, 10 ng of cDNA was used in qPCR reactions via a Bio-Rad CFX96 Real Time PCR instrument. The cDNA was then subjected to real time qPCR with standard TaqMan PCR primers and MasterMix (Applied Biosystems, Foster City, CA) for different genes. Expression of mRNA for each gene was normalized using the expression of 18S rRNA as a control endogenous gene. KO data were compared with WT data using the 2−ΔΔCt approximation method.
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