The CXCR4 mRNA level was determined using a QPCR assay. The extraction was conducted as previously described [38 (link), 39 (link)]. Briefly, Trizol buffer (Sigma-Aldrich, USA) was used to isolate cellular RNA. An Agilent Bioanalyzer (Agilent 2100; Agilent Technologies, Inc, USA) was used to assess the concentration and integrity of RNA extracted. QuantiTect Reverse Transcription Kit (QIAGEN, USA) and miRCURY LNA SYBR Green PCR Kit (QIAGEN, USA) were used to conduct RT-PCR. The protocol of PCR reaction was described previously [40 (link)]. The GAPDH gene was used as an internal reference gene to normalize the data. CXCR4 Human qPCR Primer Pair (NM_003467) and GAPDH Human qPCR Primer Pair (NM_002046) were purchased from OriGene Technologies (USA).
Trizol buffer
Manufactured by Merck Group
Sourced in United States
Trizol buffer is a monophasic solution of phenol, guanidine isothiocyanate, and other components that facilitates the isolation of total RNA from a variety of biological samples. It is commonly used in molecular biology research for the extraction and purification of RNA from cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Mircury lna sybr green pcr kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Gapdh human qpcr primer pair, by OriGene (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Hiscript q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Ncounter mouse immunology panel, by NanoString (1 mentions)
4 protocols using trizol buffer
1
CXCR4 mRNA Expression Quantification
2
RNA Extraction and qPCR for Gene Expression
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After cell collection, total RNA was extracted with Trizol buffer (Sigma-Aldrich, St. Louis, Missouri, USA). Then, the extracted RNA was converted to cDNA by reverse transcription using Hiscript QRT supermix for qPCR (+ gDNA WIPER) (Vazyme Biotech Co., Ltd, Nanjing, China) under the manufacturer’s instructions. Real-time PCR was used to detect the mRNA expression. The formula 2-∆∆Ct was used to calculate the relative mRNA expression. Primer sequences were shown in Table S3 .
3
Quantification of Immunology Transcripts
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Isolation of mRNA was performed as previously described [5 (link)]. Briefly, macrophages were lysed with TRIZOL buffer (Sigma) and total RNA was isolated by chloroform extraction and quantified (A260/A280 and A260/A230) using a nanodrop 2000 TM spectrophotometer (NanoDrop Technologies, Waltham, MA, USA). NanoString technology and the nCounter Mouse Immunology Panel (Nanostring Technologies, Seattle, WA, USA) was used to simultaneously evaluate 561 mRNAs in each sample [52 (link)]. Each sample was run in triplicate. Briefly, a total of 100 ng mRNA was hybridized to report-capture probe pairs (CodeSets) at 65 °C for 18 h. After this solution-phase hybridization, the nCounter Prep Station was used to remove excess probe, align the probe/target complexes, and immobilize these complexes in the nCounter cartridge. The nCounter cartridge was then placed in a digital analyzer for image acquisition and data processing. The expression level of each gene was measured by counting the number of times the color-coded barcode for that gene was detected, and the barcode counts tabulated. nSolver v4.0, an integrated analysis platform was used to generate appropriate data normalization as well as fold-changes, resulting ratios, and differential expression. nCounter™ v4.0 Advanced Analysis and R statistics were used to identify pathway-specific responses [52 (link)].
4
RNA Extraction from Insect Samples
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In centrifugation microtubes (Eppendorf) of 1.5 ml, 5 insects were deposited along with 1000 µl of trizol® buffer (Sigma); thereafter 3 microspheres (beads) were added to the tube. With the aid of a mechanical disruptor (Mini-Beadbeater ™), the total RNA was extracted. This done, the samples were incubated for 5 min on ice, then 200 µl of chloroform were added and the microtubes were shaken for 15 s via manual inversion, and promptly incubated for another 15 min on ice. Subsequently, the samples were centrifuged for 15 min at 4°C and 10351 xg. The next stage consisted of washing the material and for this, the supernatant was transferred to a new tube, 500 µl isopropanol was added, the solution was mixed via inversion and incubated for 10 min on ice. Further, the sample was subjected to another centrifugation for another 10 min at 4°C and 10351 xg. In this step, the supernatant was removed so that only the precipitated material (Pellet) remained and immediately 1000 µl of 75% ethanol was added, mixed via inversion for approximately 15 s and again centrifuged, this time for 5 min at 4°C and 8000 RPM.
The supernatant was again discarded and the precipitated material dried for 10 min in a laminar flow chamber. To the dry material, 35 µl of water treated with the diethyl pyrocarbonate reagent (DEPC) was added, as it is a strong inhibitor of RNase activity.
The supernatant was again discarded and the precipitated material dried for 10 min in a laminar flow chamber. To the dry material, 35 µl of water treated with the diethyl pyrocarbonate reagent (DEPC) was added, as it is a strong inhibitor of RNase activity.
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