Cells were collected and centrifugated to extract total RNA with TRlzol® reagent (Thermo Fisher Scientific). Nanodrop 2000/2000C spectrophotometry (Thermo Fisher Scientific) served to analyse the concentration and purity of the RNA. 2 μg total RNA was used for reverse transcription into cDNA by using the Promega M-MLV Reverse Transcriptase Kit in accordance with the manufacturer’s instruction. Real-PCR was carried out using SYBP Premix Ex Taq TMII. GAPDH was served as internal parameter, and 2−∆∆Ct was performed to calculate the relative expression level of each gene. The primer sequences used were listed in Additional file 1 : Table S3.
Nanodrop 2000 2000c spectrophotometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nanodrop 2000/2000C is a spectrophotometry instrument used for the analysis and quantification of nucleic acids and proteins. It utilizes a small sample volume, typically 1-2 microliters, to measure the absorbance of UV-visible light by the sample. The device provides accurate and reproducible measurements of concentrations and purity ratios.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Aceq qpcr sybr green master mix, by Vazyme (3 mentions)
Trizol reagent, by Merck Group (2 mentions)
Trlzol reagent, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase kit, by Promega (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Hiscript q rt supermix, by Vazyme (1 mentions)
Sybr green master mix, by Vazyme (1 mentions)
Hiscript q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
5 protocols using nanodrop 2000 2000c spectrophotometry
1
RNA Extraction and Quantification Protocol
2
Quantitative RT-PCR Analysis of CACYBP
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Total RNA was isolated from LNCaP and DU145 cells using TRIzol® reagent (Sigma, USA). The concentration of RNA was assessed by Nanodrop 2000/2000C spectrophotometry (Thermo Fisher Scientific). Subsequently, high-quality cDNA was synthesized with SuperScript first strand synthesis system (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Then, qRT-PCR was performed by the method using the AceQ qPCR SYBR Green master mix (Vazyme, Nanjing, China). The primer sequences were used as follows: CACYBP, forward: 5′-ACAGATCCTAGTGAGGGATTGATG-3′ and reverse: 5′-TCCGTGTCTCCTTTGGCTTG-3′; GAPDH (reference control): forward: 5′-TGACTTCAACAGCGACACCCA-3′ and reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′.
3
RNA Extraction and qPCR Analysis of Transfected Cells
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Total RNA from transfected A549 and NCI-H1299 cells was isolated using TRIzol reagent (Thermo Fisher Scientific) and the purity and integrity was assessed by Nanodrop 2000/2000C spectrophotometry (Thermo Fisher Scientific). Total RNA (1 μg) was reversely transcribed to high-quality cDNA with HiScript Q RT qPCR SuperMix kit (Vazyme). qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme). All data were normalized using GAPDH and 2−ΔΔCt method were used.
4
Transcriptional Profiling of NUBP2 Expression
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The cells were used to isolate Total RNA, by following the manufacturer's protocol, with the TRIzol ® reagent (Sigma, USA). The purity and concentration of the RNA extracted were evaluated by Nanodrop 2000/2000C spectrophotometry (Thermo, USA). Next, the cDNA was synthesized using the Hiscript QRT supermix according to the manufacturer's instructions (Vazyme, China). qRT-PCR performed using SYBR Green mastermix (Vazyme, China). For normalization purposes, GAPDH was utilized as an internal control. The 2 -ΔΔCt method was employed to determine the relative expression levels. The primers sequences (5′-3′) were listed as follows: the forward primer of NUBP2 is GTG GAG AGG CCC CAA GAA AA, the reverse primer is TAG GGA CGC AGG GCT TCT AT; the forward primer of GAPDH is TGA CTT CAA CAG CGA CAC CCA, the reverse primer is CAC CCT GTT GCT GTA GCC AAA.
5
Quantitative Gene Expression Analysis
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Total RNA was isolated from EJ and T24 cells using TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The purity and integrity of RNA was assessed by Nanodrop 2000/2000C spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (1 μg) was reversely transcribed to high-quality cDNA with HiScript Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nangjing, Jiangsu, China) according to the manufacturer's protocol. qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nangjing, Jiangsu, China). GAPDH acted as an endogenous control. Quantification of gene expression was performed using the 2-Δ ΔCt method. The primer sequences used showed in Supplementary Table 2 .
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