BM LSKs were purified from femurs and tibiae. Staining was performance as described above. Total RNA was prepared with RNA Extraction RNeasy Plus Mini- or Micro-kit (Qiagen). RNA was reverse-transcribed with High-Capacity cDNA Reverse Transcription reagents kit (Applied Biosystems) according to the manufacturer's protocol. Real-time quantitative PCR (SYBR-green, Applied Biosystems) assays were performed with an Applied Biosystems 7900HT Fast Real-Time PCR System sequencer detector. Expression was normalized to Hprt (F-5′-CCTAAGATGAGCGCAAGTTGAA-3′, R-5′-CCACAGGACTAGAACACCTGCTAA-3′) and 36b4 (F-5′-ACTGGTCTAGGACCCGAGAAG-3′, R-5′-TCCCACCTTGTCTCCAGTCT-3′) expression. The primer sequences for Glg-1 were: F-5′-CAAGATGACGGCCATCATTTT-3′, R-5′-TTCCCCAAGACGAATGCTGC-3′; for p18: F-5′-TTATGAAGCACACAGCCTGCAATGT-3′, R-5′-ACGGACAGCCAACCAACTAACGG-3′; for p21: F-5′-TGTCTTGCACTCTGGTGTCTGAGC-3′, R-5′-TCTTGCAGAAGACCAATCTGCG-3′; for p57: F-5′-GCGCAAACGTCTGAGATGAG-3′, R-5′-AGAGTTCTTCCATCGTCCGCT-3′; for Tgfb1: F-5′-CCGAAGCGGACTACTAT-3′; R-5′-GTAACGCCAGGAATTGT-3′.
7900ht fast real time pcr system sequencer detector
Manufactured by Thermo Fisher Scientific
The 7900HT Fast Real-Time PCR System is a high-performance quantitative PCR (qPCR) instrument designed for fast, accurate, and sensitive detection and quantification of nucleic acids. It features a high-resolution CCD detector, a fast thermal cycler, and flexible software for a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Thermo Fisher Scientific (4 mentions)
Rna extraction rneasy plus mini or micro kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription reagents kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Merck Group (2 mentions)
Rneasy plus mini rna extraction kit, by Qiagen (1 mentions)
4 protocols using 7900ht fast real time pcr system sequencer detector
1
Quantitative PCR Analysis of LSK Cell Markers
2
Quantitative Transcriptional Profiling of Progenitor Cells
3
Quantitative Real-Time PCR Analysis
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Total RNA from sorted macrophages was prepared with RNA Extraction RNeasy Plus Micro-kit (QIAGEN). For whole tissue RNA extraction, TRIzol (Sigma-Aldrich) extraction method was used. RNA was reverse-transcribed with High-Capacity cDNA Reverse Transcription reagents kit (Applied Biosystems) according to the manufacturer’s protocol. Real-time quantitative PCR (SYBRGreen; Applied Biosystems) assays were performed with an Applied Biosystems 7900HT Fast Real-Time PCR System sequencer detector. Expression was normalized to 36b4 expression. Primer sequences are listed in the Table S2.
4
RNA Extraction and Real-Time qPCR
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Total RNA was prepared with RNA Extraction RNeasy Plus Mini- or Micro-kit (QIAGEN) for isolation from macrophages or with TRIzol (Sigma) for total tissue RNA. RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s protocol. Real-time quantitative PCR (SYBR-green; Applied Biosystems) assays were performed with an Applied Biosystems 7900HT Fast Real-Time PCR System sequencer detector. Expression was normalized to Hprt and/or 36b4 expression. Primer sequences are listed in Table S3.
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