Total RNA was extracted from samples using TRIzol reagent (Life technologies) and reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative reverse transcription-PCR (qRT–PCR) was performed to detect mRNA expression using Taqman Universal Mix II (Applied Biosystems) with Taqman probes (Applied Biosystems, Mm01731290_g1 for p53, Mm00432448_m1 for Cdkn1a, Mm00449846_m1 for Phlda3, Mm01281680_m1 for Lmo2, Mm01342805_m1 for Hes1 and Mm99999915_g1 for Gapdh). Gapdh was used as an internal control to correct for the concentration of RNA in different samples. Expression of miR-30-based p53.1224 shRNA was detected using a Custom TaqMan MicroRNA Assay (Applied Biosystems). A reverse transcription primer and a TaqMan probe were designed to specifically detect p53.1224 siRNA56 (link). In vitro reverse transcription was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). SnoRNA 202 was used as an internal control to correct for the concentration of RNA in different samples. Each experiment was performed with three replicates from each sample, and the results were averaged.
Taqman universal mix 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Taqman Universal Mix II is a ready-to-use, optimized reaction mix designed for real-time PCR applications. It contains the necessary components for efficient DNA amplification and detection, including a thermostable DNA polymerase, dNTPs, MgCl2, and a proprietary buffer system.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Custom taqman microrna assay, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Taqman validated assays, by Thermo Fisher Scientific (1 mentions)
Mirneasy, by Qiagen (1 mentions)
Abi prism 7500 sequence detector, by Thermo Fisher Scientific (1 mentions)
Mir 106b 5p, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman universal mix 2
1
Quantifying mRNA and miRNA Expression
2
Quantitative analysis of p53-related gene expression
3
Quantifying miR-34 Family Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was purified by miRNeasy (Qiagen, Germantown, MD, USA) and reverse-transcribed by TaqMan Universal Mix II (Applied Biosystems, Waltham, MA, USA) using miRNA-specific assay reverse transcription. Semiquantitative PCR was performed with TaqMan-validated assays (Applied Biosystems): miR34a (000426), hsa-miR34b (000427), miR34c (000428), and hsa-miR34c-3p (241009_mat). As reference for cDNA, we chose U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real-time data were collected using Microsoft Excel and analyzed with the following formula: expression level = 2-ΔΔCt method. All experiments were done as independent triplicates and analyzed using standard deviation (SD). The p-value was obtained with the Student’s t-test.
4
Plasma miRNA and SP-D Expression Analysis in COPD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from plasma using TRIzol reagent (Ambion; Thermofisher Scientific, Inc.). Total RNA obtained from plasma was transcribed to cDNA using the TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.), and qRT-PCR amplification with TaqMan™ Universal MixII (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.). U6 was used as an internal control. All primers (U6, miR-486-5p, miR-106b-5p) corresponding to miRNAs were bought from Applied Biosystems (Thermo Fisher Scientific, Inc. Cat. No. 4427975, 4427975, 4427975). The expression of SP-D were detected by SYBR Green system and normalized with β-actin. The primers were as follows: SP-D, sense 5’-GGGAGAAGATTTTCAAGACAGC-3’ and antisense 5’-CCTCTGTCTTGGAATCAGTCAT-3’; β-actin, sense 5’-GCGGGAAATCGTGCGTGAC-3’ and antisense 5’-GGAAGGAAGGCTGGAAGAG -3’; qRT-PCR analysis was performed using an ABI Prism 7500 Sequence Detector (Applied Biosystems, FosterCity, CA, USA), and calibrated by using the 2-ΔΔCT method.
ELISA analysis Plasma of Tibetan healthy people and COPD patients were subjected to ELISA analysis for their concentration of SP-D. SP-D ELISA kits from Bioswamp (Wuhan, Hubei, China) were used according to the manufacturer's instructions.
ELISA analysis Plasma of Tibetan healthy people and COPD patients were subjected to ELISA analysis for their concentration of SP-D. SP-D ELISA kits from Bioswamp (Wuhan, Hubei, China) were used according to the manufacturer's instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!