Total RNA was extracted from in vitro cultured cells using a Qiagen RNeasy Mini kit (Qiagen, Redwood City, CA, USA) according to the manifacturer’s instructions. Reverse transcription was performed using RevertAid Reverse Transcriptase (Thermoscientific). qRT-PCR was performed with a Gene-Amp 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA), using the SYBR green dye detection method. The mRNA levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers used to analyze each gene were: HIF-1α (Fw:CCAGTTAGGTTCCTTCGATCAGT, Rv:TTTGAGGACTTGCGCTTTCA); BMI1 (Fw:ATGTGTGTGCTTTGTGAG, Rv:AGTGGTCTGGTCTTGTGAAC); SOX2 (Fw:CACCCCTGGCATGGCTCTT, Rv:GAGCTGGCCTCGGACTTGA); NANOG (Fw:AATACCTCAGCCTCCAGCAGATG, Rv:TGCGTCACACCATTGCTATTCTTC); OCT4 (Fw:TCCCATGCATTCAAACTGAGGT, Rv:CCCAAAAACCCTGGCACAA); GAPDH (Fw:TCCTGAGCTGAACGGGAAG, Rv:GGAGGAGTGGGTGTCGCTGT); PUMA (Fw: AAGTCAGGACTTGCAGGCGCG, Rv: TGGGTCCCAGTCAGTGTGTGT); NOXA (Fw: CGCTGACGACGTCCCAGCGTTT, Rv: CGAAGACGGCGTTATGGGAGC); BIM (Fw: CAGAGATATCGATCGCCCAAG, Rv: CAGAGATATGGATCGCCCAAG); BCl-2 (Fw:CTGCACCTGACGCCCTTCACC, Rv: CACATGACCCCACCGAACTCAAAGA); BAX (Fw: TCCCGGCTCTCTGATCCCCG, Rv: GGCTAGGGGAACGCTATATGC); BCL-XL (Fw: TTGGATGGCCACTTACCTGAAT, Rv: AACCAGCGGTTGAAGCGTT). Student’s t-test was used and results were considered to be statistically significant if p < 0.05 (*).
Geneamp 7900
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneAmp 7900 is a real-time PCR system designed for quantitative gene expression analysis. It provides precise and reliable detection and quantification of nucleic acid sequences. The GeneAmp 7900 utilizes a high-performance optical system and advanced thermal cycling technology to enable accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
Superscript 4 vilo system, by Thermo Fisher Scientific (1 mentions)
Power sybr green, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Power sybr green chemistry, by Thermo Fisher Scientific (1 mentions)
6 protocols using geneamp 7900
1
Quantitative Analysis of Stem Cell Markers
2
Placental Gene Expression Analysis
3
Genetic Analysis of FOXP3 Microsatellite in Allo-SCT
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Donor and recipient genomic DNA was purified from EDTA anticoagulated peripheral blood before allo-SCT. Genotyping of the (GT)n microsatellite polymorphism in the FOXP3 gene was performed by a fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) method (GeneAmp 7900; Applied Biosystems) and sized by capillary electrophoresis (POP7—ABI PRISM 3130 xL Genetic Analyzer; Applied Biosystems) and fragment analysis (GeneMapper 4.0 Software; Applied Biosystems) as previously described [21 (link)]. FOXP3 alleles were divided in two groups: short alleles (with 15 or less microsatellite repeats; ≤(GT)15) and long alleles (with 16 or more microsatellite repeats; ≥(GT)16) [22 (link)]. Hemizygous individuals were included in their respective homozygous genotype group [27 (link)]. As suggested by Engela et al. [22 (link)], short/long heterozygous females were included in the short allele group.
4
Hypoxia and Estrogen Regulation of Erythroid Genes
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Erythroid cells at the EB stage were used after 1, 2, or 3 weeks in culture under hypoxia (with and without estrogen). RNA was isolated from EBs using RNeasy Mini Kit (Qiagen). Complementary DNA was produced from total RNA through RT-PCR using a Superscript Vilo IV System (Invitrogen). Real-time PCR was performed using a GeneAmp 7900 sequence detection system with POWER SYBR Green (Applied Biosystems). The primer sequences used were as follows:
GATA1-L, 5′-CCTGCTTTGTTGCCAATG-3′ and GATA1-R, 5′-CTGCTCCACT GTTACGGATAC-3′; VEGF-L, 5′-ATCTTCAAGCCATCCTGTGTGC-3′ and VEGF-R, 5′-CAAGGCCCACAGGGATTTTC-3′; EpoR-L, 5′-GCACCGAGTGTGTGCTGAGCAA-3′ and EpoR-R, 5′-GGTCAGCAGCACCAGGATGAC-3′; ALAS-2-L, 5′-ACAGTGCTGCCCAGTGCTTG-3′ and ALAS-2-R, 5′-TCCGACAGCATGAAGGGACA-3′; HIF1A-L, 5′-GAAAGCGCAAGTCTTCAAAG-3′ and HIF1A-R, 5′-TGGGTAGGAGATGGAGATGC-3′; HIF1B-L, 5′- CCCCGAAATGACATCAGATG-3′ and HIF1B-R, 5′-GTTCCCTTCTCCATCATCATC-3′; Bcl-xL-L, 5′-GCAGGTATTGGTGAGTCGGATCGC-3′ and Bcl-xL-R, 5′-CACAAAAGTATCCCAGCCGCCG-3′. The expression level of GAPDH was used to normalize the results, with the following primers: GAPDH-L, 5′-CTGGCATTGCCCTCAACGACC-3′ and GAPDH-R, 5′-CTTGCTGGGGCTGGTGGTCC-3′.
GATA1-L, 5′-CCTGCTTTGTTGCCAATG-3′ and GATA1-R, 5′-CTGCTCCACT GTTACGGATAC-3′; VEGF-L, 5′-ATCTTCAAGCCATCCTGTGTGC-3′ and VEGF-R, 5′-CAAGGCCCACAGGGATTTTC-3′; EpoR-L, 5′-GCACCGAGTGTGTGCTGAGCAA-3′ and EpoR-R, 5′-GGTCAGCAGCACCAGGATGAC-3′; ALAS-2-L, 5′-ACAGTGCTGCCCAGTGCTTG-3′ and ALAS-2-R, 5′-TCCGACAGCATGAAGGGACA-3′; HIF1A-L, 5′-GAAAGCGCAAGTCTTCAAAG-3′ and HIF1A-R, 5′-TGGGTAGGAGATGGAGATGC-3′; HIF1B-L, 5′- CCCCGAAATGACATCAGATG-3′ and HIF1B-R, 5′-GTTCCCTTCTCCATCATCATC-3′; Bcl-xL-L, 5′-GCAGGTATTGGTGAGTCGGATCGC-3′ and Bcl-xL-R, 5′-CACAAAAGTATCCCAGCCGCCG-3′. The expression level of GAPDH was used to normalize the results, with the following primers: GAPDH-L, 5′-CTGGCATTGCCCTCAACGACC-3′ and GAPDH-R, 5′-CTTGCTGGGGCTGGTGGTCC-3′.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cultured cells using a Qiagen RNeasy Mini kit (Qiagen, Redwood City, CA, USA) according to the manifacturer’s instructions. Reverse transcription was performed using RevertAid Reverse Transcriptase (Thermo Scientific). qRT-PCR was performed with a Gene-Amp 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA), using the SYBR green dye detection method. The mRNA levels were normalized using β-actin (ACTB) transcript. Relative mRNA levels were measured using the 2-Δ cycle threshold (2-ΔCT) method. Primers used to analyze each gene were: Sema5A: 5’-ACTGTTCTAGCGACGGCACC-3′ (forward), 5’-CCCCAGAAAGCCCATCTGT-3′(reverse), c-Myb: 5’-AAGTCTGGAAAGCGTCACTTG-3′ (forward), 5’-ACATCTGTTCGATTCGGGAGATA-3′ (reverse), β-actin: 5’-ATTGCCGACAGGATGCAGAA-3′ (forward), 5’-GCTGATCCACATCTGCTGGAA-3′ (reverse). Student’s t-test was used and results were considered significant if p < 0.05.
6
Hypoxia-Induced Transcriptional Regulation
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EBs were extracted from the culture media after weeks 1, 2, and 3 in hypoxia. RNA was isolated from EB using an RNeasy Mini kit (QIAGEN). cDNA was produced from total RNA through RT-PCR using a Superscript III First-Strand Synthesis system (Invitrogen). Real-time PCR was performed using a GeneAmp 7900 sequence detection system using POWER SYBR Green chemistry (Applied Biosystems). The primer sequences were as follows: VEGF165-L, 5′-ATCTTCAAGCCATCCTGTGTGC-3′; VEGF165-R, 5′-CAAGGCCCACAGGGATTTTC-3′; Vegf-A(all isoforms)F, 5′-GAGATGAGCTTCCTACAGCAC-3′; Vegf-A(all isoforms)R, 5′-TCACCGCCTCGGCTTGTCACAT-3′; FLT3-F, 5′-TTTCACAGGACTTGGACAGAGATTT-3′; FLT3-R, 5′-GAGTCCGGGTGTATCTGAACTTCT-3′; TPO-F, 5′-CAGGACTGAAAAGGGAATCA-3′; TPO-R, 5′-CGTTGGAAGGCCTTGAATTT-3′; GATA1-L, 5′-CCTGCTTTGTTGCCAATG-3′; GATA1-R, 5′-CTGCTCCACTGTTACGGATAC-3′; Bcl-xL-L, 5′-GCAGGTATTGGTGAGTCGGATCGC-3′; and Bcl-xL-R, 5′-CACAAAAGTATCCCAGCCGCCG-3′. The expression level of GADPH was used to normalize the results. GADPH-L, 5′-CTGGCATTGCCCTCAACGACC-3′ and GADPH-R, 5′-CTTGCTGGGGCTGGTGGTCC-3′.
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