ATMs were lysed in Qiazol and total RNA was purified using Qiagen miRNeasy Micro kit. RNA concentration and purity was measured using a NanoDrop 2000 spectrophotometer. 400ng total RNA was reverse transcribed to cDNA using Qiagen miScript II RT kit with HiFlex buffer. To validate miRNA expression by qRT-PCR, miScript SYBR Green PCR kit and miScript miRNA Primer Assays were used (Qiagen).
Miscript mirna primer assays
Manufactured by Qiagen
Sourced in United States
The MiScript miRNA Primer Assays are lab equipment used for the detection and quantification of microRNA (miRNA) expression. The assays provide a reliable and sensitive method for analyzing miRNA levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Miscript sybr green pcr kit, by Qiagen (3 mentions)
Miscript 2 rt kit, by Qiagen (3 mentions)
Mirneasy micro kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green, by Bio-Rad (1 mentions)
Nanodrop 2000 spectrophotometer, by Agilent Technologies (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Miscript 2 rt, by Qiagen (1 mentions)
4 protocols using miscript mirna primer assays
1
miRNA Expression Profiling from ATMs
2
Analyzing miRNA Expression in ATM Samples
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ATMs were lysed in Qiazol and total RNA was purified with Qiagen miRNeasy Micro/Mini kit. RNA concentration and purity was measured using a NanoDrop 2000 spectrophotometer or Agilent Bioanalyzer. RNA was reverse transcribed to cDNA with the miScript II RT kit (Qiagen). Quantitative RT-PCR was performed with miScript SYBR Green PCR kit (Qiagen) or SSO Advanced Universal SYBR Green (BioRad). MiScript miRNA Primer Assays (Qiagen) were used for miRNA qRT-PCR. Genes were amplified with the following primers purchased from Integrated DNA Technologies—Beta-Actin: Fwd-GGCTGTATTCCCCTCCATCG, Rev-CCAGTTGGTAACAATGCCATGT; Tnfa: Fwd-CTGAACTTCGGGGTGATCGG, Rev-GGCTTGTCACTCGAATTTTGAGA; IL6: Fwd-CCAAGAGGTGAGTGCTTCCC, Rev-CTGTTGTTCAGACTCTCTCCCT; Ccl2: Fwd-TTAAAAACCTGGATCGGAACCAA, Rev-GCATTAGCTTCAGATTTACGGGT; Ccl3: Fwd-TTCTCTGTACCATGACACTCTGC, Rev-CGTGGAATCTTCCGGCTGTAG. Fold change in expression was determined by the comparative cycle method (2−ΔΔCt).
3
miRNA Expression Profiling from ATMs
4
Quantification of miR-34a and Targets
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Expression levels of miR-34a and its target genes were assessed by RT-qPCR following treatment with miR/NG polyplexes. U-87 MG GBM cells (3×10 ^5 cells /well) were seeded in 10 cm plates (37°C; 5% CO2). Twenty-four hours later U-87 MG cells were transfected with 200 nM miR-34a or NC-miR complexed with the indicated NGs [NG3/miR-34a and NG4/miR-34a both N/P=5] or left untreated. Total RNA was extracted (EZ-RNA II, Biological Industries) from each sample and subjected to reverse transcription into cDNA (miSCript II RT, Qiagen). Samples were evaluated for the expression levels of miR-34a and its target genes using SYBR green real-time qPCR (StepOne plus, Life Technologies). miR-34a expression levels were evaluated by miSCript miRNA primer assays (Qiagen, USA) according to the manufacturer's protocol and were normalized to RNU6. Expression levels of miR-34a target genes were assessed using custom made primers (Syntezza Bioscience, Israel) and SensiFAST TM SYBR green (Bioline, United Kingdom) according to manufacturer's protocol, and were normalized to Actin. Target genes primers pairs were:
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