RediPlate™ 96 RiboGreen™ RNA Quantitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Quantitation Range is 3-200 ng	Add 180 µL of the provided RediPlate TE buffer (Component B) to samples. Incubate samples for 10 minutes at room temperature, protected from light.

Upstream tips
Quantitation Range is 3-200 ng
Protocol tips
Add 180 µL of the provided RediPlate TE buffer (Component B) to samples. Incubate samples for 10 minutes at room temperature, protected from light.

Publication protocol

At the end of the treatment period, total RNA was isolated from LS180 cells using TRIzol and reverse transcribed using Superscript II reverse transcriptase, as described previously (Chang et al., 2006). Total RNA concentration and total cDNA concentration were quantified by the RiboGreen RNA Quantitation Kit and PicoGreen dsDNA Quantitation Kit, respectively (Chang et al., 2006). Polymerase chain reaction (PCR) was performed in a real-time DNA thermal cycler (LightCycler; Roche Diagnostics). Each 20-μl PCR reaction contained 1 U of Platinum Taq DNA polymerase in 1× PCR II buffer (20 mM Tris-HCl, pH 8.4, and 50 mM KCl), 3 mM MgCl2 (except that 4 mM was used in the amplification of CYP3A4 cDNA), 0.25 mg/ml bovine serum albumin, 0.2 mM dNTP, 0.2 μM forward and reverse primers specific for CYP3A4, CYP3A5, and ABCB1, 1:30,000 SYBR Green I, and 10 ng of total cDNA. The sequences of the forward and reverse primers to amplify human CYP3A4 cDNA were 5′-CCT-TAC-ACA-TAC-ACA-CCC-TTT-GGA-AGT-3′ and 5′-AGC-TCA-ATG-CAT-GTA-CAG-AAT-CCC-CGG-TTA-3′, respectively (Schuetz et al., 1996). The sequences of the forward and reverse primers to amplify human CYP3A5 cDNA were 5′-CCT-TAC-ATA-TAC-ACA-CCC-TTT-GGA-AC-3′ and 5′-GTT-GAA-GAA-GTC-CTT-GCG-TGT-C-3′, respectively (Yamaori et al., 2005). The sequences of the forward and reverse primers to amplify human ABCB1 cDNA were 5′-GGC-CTA-ATG-CCG-AAC-ACA-TT-3′ and 5′-CAG-CGT-CTG-GCC-CTT-CTT-C-3′, respectively (Liu et al., 2002). The sequences of the forward and reverse primers to amplify human PXR cDNA were 5′-CAA-GCG-GAA-GAA-AAG-TGA-ACG-3′ and 5′-CAC-AGA-TCT-TTC-CGG-ACC-TG-3′, respectively (Chang et al., 2003). The cycling conditions for the real-time amplification (LightCycler; Roche Diagnostics) were 94°C for 5 s (denaturation), 60°C for 10 s (annealing), and 72°C for 15 s (extension) for CYP3A4, 95°C for 1 s (denaturation), 65°C for 6 s (annealing), and 72°C for 10 s (extension) for CYP3A5, 95°C for 1 s (denaturation), 61°C for 6 s (annealing), and 72°C for 10 s (extension) for ABCB1, and 95°C for 5 s (denaturation), 65°C for 10 s (annealing), and 72°C for 15 s (extension) for PXR. The initial denaturation was performed at 95°C for 5 min. Calibration curves were constructed by plotting the cross-point against known amounts of purified amplicon, as determined by the PicoGreen dsDNA Quantitation Kit. Initial experiments were performed to verify the specificity of the primers by purifying and sequencing the amplicons. Purification was carried out using the QIAquick Gel Extraction Kit, according to the manufacturer's instructions (QIAGEN, Valencia, CA). Purified amplicons were sequenced using the Applied Biosystems 377 DNA sequencer (Applied Biosystems, Inc., Foster City, CA) at the Nucleic Acid and Protein Service Unit at the University of British Columbia. The identity of the purified amplicons was confirmed using the BLAST program (http://www.ncbi.nlm.nih.gov).

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for RediPlate™ 96 RiboGreen™ RNA Quantitation Kit below.

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