To validate identified miRNAs with statistical significance, qPCR analysis of selected miRNA targets was performed. Applied Biological Materials Inc. (ABM, BC, Canada). First, 50 ng of exosomal total RNA was reverse transcribed with the miRNA cDNA synthesis kit, with Poly (A) polymerase tailing (ABM, BC, Canada) according to the manufacturer’s instructions. Then, equal amounts of cDNA were used for all the qPCR reactions. qPCR analysis was conducted with an Applied Biosystems 7900HT thermal cycler using the Fast Advanced Master Mix (Applied Biosystems). For 351 miRNAs, a large transit analysis was performed, and then for a selected few of miRNAs (such as hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-182-5p, hsa-miR-200c-3p, hsa-miR-204-5p, hsa-miR-205-5p, hsa-miR-221-3p, hsa-miR-301a-3p, hsa-miR-375, hsa-miR-451a, hsa-miR-455-3p, hsa-miR-500a-5p, hsa-miR-503, hsa-miR-542-5p, hsa-miR-582-5p) the TaqMan Advanced miRNA Assays were used. The levels of differentially expressed miRNAs isolated from uEVs from patients with diabetic nephropathy were compared to the control subjects. The miRNA expression fold change (FC) was expressed as base-2 logarithm of FC (log2FC) to normalize the miRNA expression values.
7900ht thermal cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900HT thermal cycler is a high-performance instrument designed for real-time PCR applications. It features precise temperature control and fast ramp rates to facilitate efficient DNA amplification. The 7900HT is capable of running a wide range of PCR protocols and supports multiple sample formats.
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Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Lenti x qrt pcr kit, by Takara Bio (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Sds 2, by Thermo Fisher Scientific (3 mentions)
Calphos transfection kit, by Takara Bio (3 mentions)
Ppackh1 lentivector packaging mix, by System Biosciences (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
High capacity rna cdna kit, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hek293ft cells, by Thermo Fisher Scientific (2 mentions)
Mirna cdna synthesis kit, by Applied Biological Materials (1 mentions)
Fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Peqgold rna kit, by Avantor (1 mentions)
Sybr green mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 detection system, by Bio-Rad (1 mentions)
Data assist software ver 3, by Thermo Fisher Scientific (1 mentions)
30 protocols using 7900ht thermal cycler
1
Validating miRNA Targets via qPCR
2
Hypoxia-Induced Changes in mRNA Expression
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Tumor cells were seeded at 104 cells/well in 48 well plates in DMEM with 10% FBS. The cells were cultivated at either 20% or 2% oxygen (5% CO2) for 48 h and harvested by passive lysis. RNA was isolated using the PeqGold RNA kit (Peqlab, Erlangen, Germany) according to the manufactures instructions.
mRNA-Expression was quantified by qRT-PCR on an 7900HT Thermal cycler (Applied Biosystems, Darmstadt, Germany) using SYBR green mix (Fermentas, Darmstadt, Germany). Sequences of primers used in qRT-PCR are given insupplemental table 1 . All primer pairs were designed to span at least one intron to avoid amplification of contaminating gDNA. Expression levels of lox-family members were normalized against expression of rsp2960 (link).
mRNA-Expression was quantified by qRT-PCR on an 7900HT Thermal cycler (Applied Biosystems, Darmstadt, Germany) using SYBR green mix (Fermentas, Darmstadt, Germany). Sequences of primers used in qRT-PCR are given in
3
Profiling Exosomal miRNA Expression
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miRNA in ExoHypoxic and ExoNormoxic were analyzed using TaqMan® Array MicroRNA Cards with pre-amplification kit (Life Technologies, Applied Biosystems). 350 ng of miRNA was used as input in each reverse transcription (RT) reaction. The RT reaction and pre-amplification steps were done according to the manufacturer’s instructions. miRNAs were reverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies, Applied Biosystems). RT reaction products from the exosome sample were further amplified using the Custom PreAmp Primer Pool. The expression profile of miRNAs was determined using the TaqMan® Universal Master Mix II (Life Technologies, Applied Biosystems) in an Applied Biosystems 7900HT thermal cycler using the manufacturer’s recommended program. Finally, all the raw data from each array were retrieved from the 7900HT and run on Data Assist Software ver.3.1 (Applied Biosystems). In another set of experiments, we collected exosomes from the conditioned medium of RWPE1, LNCaP, PC-3 and DU145 cells; RNA was isolated from cells and exosomes, followed by miRNA cDNA synthesis (Applied Biological Materials Inc., BC, Canada). Real-time PCR was performed using SYBR Green master mix on a Bio-Rad CFX96 detection system (Bio-Rad, Hercules, CA). The fold change in expression levels was determined using RNU6 and 5S rRNA as an internal control.
4
Quantitative Analysis of miR-206 Expression
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Quantitative real-time PCR (qRT-PCR) was used to determine the miR-206 expression. The primers for RT-PCR were designed based on the miR-206 sequences provided by the Sanger Center miRNA Registry. The RT primers were designed as follows: RT primer: CTC AGC GGC TGT CGT GGA CTG CGC GCT GCC GCT GAG CCA CAC AC. PCR primers: F: GGC GGT GGA ATG TAA GGA AG; R: GGC TGT CGT GGA CTG CG. The qRT-PCR was performed on the ABI (Applied Biosystems) 7900 HT Thermal cycler in standard mode for 40 cycles. Relative miRNA expression values (Target miRNA vs U6) were calculated with the 2 −ΔΔCt method.
5
Relative Telomere Length Quantification
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Genomic DNA was extracted directly from H-ASCs and AAA-ASCs using standard procedures. The relative telomere length was determined as previously described [21 (link)]. Briefly, the telomere length was represented by the relative ratio of the telomere repeat copy number (T) to the single-copy gene 36B4 copy number (S). The T/S ratio was determined by quantitative polymerase chain reaction (qPCR) using a 7900HT thermal cycler (Applied Biosystems). It can then be calculated by the formula T/S = 2(−dCt), where dCt is the difference in threshold cycle obtained by subtracting the average 36B4 Ct value from the average telomere Ct value. Primer sequences are as follows: Tel 1b—270 nM, 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′; Tel 2b—900 nM, 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′, 36B4u—300 nM, 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; and 36B4d—500 nM, 5′-CCCATTCTATCATCAACGGGTACAA-3′. All samples were measured in triplicate.
6
Quantitative miRNA Expression Analysis
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Equal amounts of RNA were reverse transcribed with the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions, with a custom 1X RT primer pool (hsa-miR-101–3p ID 002253; U6 snRNA ID 001973; hsa-miR-424 ID 000604; hsa-miR-34a ID 000426; hsa-miR-21 ID 000397; hsa-miR-504 ID 002084; hsa-miR-92a ID 000431). Real time PCR analysis was performed with an Applied Biosystems 7900HT thermal cycler using 20X Individual TaqMan® MicroRNA Assays.
7
Identifying Secreted Chemoattractants in Glioblastoma Stem Cells
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To identify potential chemoattractants differentially expressed by GSCs, we used qPCR array (RT-qPCR analysis) to determine the expression of major secreted proteins, including most cytokines along with some secreted proteins relevant to tumorigenesis, in paired GSCs and NSTCs. qPCR primers were designed to span an intron of each target gene (Supplementary Table 1 ). Total mRNA was purified from paired GSCs and NSTCs with the RNeasy Mini Kit (74104, Qiagen). 500 ng mRNA was reverse transcribed into cDNA with SuperScript III Reverse Transcriptase (18080-044, Invitrogen). RT-qPCR assays were performed with SYBR Green PCR Master Mix (4310251, Applied Biosystems) on a 7900HT thermal cycler (Applied Biosystems). Three-step amplification was performed (95°C 15 sec, 60°C 15 sec, and 72°C 30 sec) for 45 cycles. For data analysis, GAPDH was used as the internal standard and fold changes of gene expression levels in GSCs relative to NSTCs were calculated. The target genes with a > 2 fold upregulation in GSCs were regarded as differentially expressed. RT-qPCR arrays were performed with 3 pairs of matched GSCs and NSTCs and repeated for three times. Average fold changes of target genes presented were from 3 representative experiments.
8
Quantifying Bcl-x Expression Changes
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Total RNA was extracted from paired untreated and tamoxifen-treated Eμ-Myc lymphoma cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was treated with DNase to remove contaminating DNA using the RNase-free DNase Qiagen kit according to the manufacturer's instructions. RNA quality and quantity were determined using the NanoDrop assay (Thermo Fisher Scientific). Aliquots of 1 μg of RNA were reverse-transcribed into cDNA using the SuperScript III first strand synthesis Supermix kit (Invitrogen) in a 20-μL reaction volume according to the manufacturer's instructions. The cDNA was diluted 10-fold in H2O, and PCR amplifications of 1 μL of cDNA were performed with an Applied Biosystems 7900HT thermal cycler using TaqMan Universal PCR Mastermix and 0.5 μL of TaqMan primer/probes (both Applied Biosystems) in a 10-μL reaction volume. Assays specific for Bcl-x (Mm00437783) and (as an endogenous control for RNA quality/input) HMBS (Mm01143545) transcripts were performed. Three replicates of each reaction were performed. All qPCR data were analyzed using the 2−ΔΔCT method and expressed relative to the untreated sample of each pair.
9
Quantitative gene expression analysis
10
Quantitative Profiling of Germ Layer Markers
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Total RNA was isolated from EBs (n = 6, biological replicates) using an RNeasy Mini Kit (Qiagen) and treated with DNase I according to the manufacturer’s instructions. Synthesis of cDNA was performed using the High Capacity RNA-to-cDNA Kit (Qiagen) with 1 µg total RNA per the manufacturer’s instructions. A TaqMan array card (Applied Biosystems) containing primers and probes was dried in a 384-well plate. We curated sets of marker genes for each of the three germ layers (Supplementary Data 2 ). TaqMan 2× Universal PCR master mix (Applied Biosystems) was used for PCR analysis, which was carried out on an Applied Biosystems 7900HT thermal cycler under the following cycling conditions: incubation at 50 °C for 2 min and 10 min of denaturation at 94.5 °C, followed by 40 cycles of 97 °C for 30 s and 60 °C for 1 min. Each gene was run in duplicate. Gene-expression levels were calculated using the comparative Ct method with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the endogenous housekeeping gene, and results were normalized to the average of the 201B7 samples to obtain fold changes. After data standardization (z-scoring) of each of the three germ layer marker genes, PCA was performed using SYSTAT 13 Software (Systat Software Inc.). The first principal component score (PC1) was calculated for further analysis.
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