RNA, DNA, and Protein from a Single Sample
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TRI Reagent® (patented product/reagent) is a ready-to-use reagent for the isolation of total RNA or the simultaneous isolation of RNA, DNA, and protein from diverse biological samples. During a one-step sample homogenization/ lysis procedure, TRI Reagent disrupts cells and denatures endogenous nucleases. The homogenate is separated into aqueous and organic phases by addition of bromochloropropane (or chloroform) followed by centrifugation (see sidebar, Advantages of Using Bromochloropropane Instead of Chloroform). RNA partitions to the aqueous phase, DNA to the interphase, and protein to the organic phase.
This highly reliable technique performs well with both small and large quantities of tissues and cultured cells (e.g., samples larger than ~5 mg tissue or 5 x 105 cultured cells) (Figure 1). RNA isolated using TRI Reagent is suitable for use in real-time and end-point RT-PCR, amplification/labeling for microarray analyses, in vitro translation, cDNA synthesis, Northern blotting, and RNase protection assays. RNA preparations isolated using bromochloropropane often do not require an additional DNase treatment, but Ambion's TURBO DNA-free Kit can be used to remove residual genomic DNA before RT-PCR assays. In addition, PCR, Southern blotting, and cloning can be performed using DNA, and denaturing polyacrylamide gel electrophoresis, and Western blot assays can be performed using protein isolated with TRI Reagent.
Figure 1. Bromochloropropane (Phase Separation Reagent) Improves Genomic DNA Removal in the TRI Reagent® Single-Step RNA Isolation Method. Total RNA was isolated from RNAlater®-treated mouse liver, kidney, brain, lung, heart, and spleen and from fresh cultured human cells using the standard TRI reagent protocol with either bromochloropropane (BCP) or chloroform (CHCl3).(A) Genomic DNA removal was assessed by one-step, (+) or (–) RT, quantitative PCR targeting mouse or human TATA binding protein (TBP) mRNA and gene sequences, and is presented as percent of residual gDNA. (B) Total RNA yield was determined by A260 using the NanoDrop® spectrophotometer, and RNA yield (µg/mg) was normalized to mg of mouse tissue input or to 0.5 x 105 human cultured cells.
Scientific Contributors
Quoc Hoang, WeiWei Xu, Roy Chris Willis, Angela Burrell, Mangkey Bounpheng, Xingwang Fang • Ambion, Inc.
This highly reliable technique performs well with both small and large quantities of tissues and cultured cells (e.g., samples larger than ~5 mg tissue or 5 x 105 cultured cells) (Figure 1). RNA isolated using TRI Reagent is suitable for use in real-time and end-point RT-PCR, amplification/labeling for microarray analyses, in vitro translation, cDNA synthesis, Northern blotting, and RNase protection assays. RNA preparations isolated using bromochloropropane often do not require an additional DNase treatment, but Ambion's TURBO DNA-free Kit can be used to remove residual genomic DNA before RT-PCR assays. In addition, PCR, Southern blotting, and cloning can be performed using DNA, and denaturing polyacrylamide gel electrophoresis, and Western blot assays can be performed using protein isolated with TRI Reagent.
Figure 1. Bromochloropropane (Phase Separation Reagent) Improves Genomic DNA Removal in the TRI Reagent® Single-Step RNA Isolation Method. Total RNA was isolated from RNAlater®-treated mouse liver, kidney, brain, lung, heart, and spleen and from fresh cultured human cells using the standard TRI reagent protocol with either bromochloropropane (BCP) or chloroform (CHCl3).(A) Genomic DNA removal was assessed by one-step, (+) or (–) RT, quantitative PCR targeting mouse or human TATA binding protein (TBP) mRNA and gene sequences, and is presented as percent of residual gDNA. (B) Total RNA yield was determined by A260 using the NanoDrop® spectrophotometer, and RNA yield (µg/mg) was normalized to mg of mouse tissue input or to 0.5 x 105 human cultured cells.
Scientific Contributors
Quoc Hoang, WeiWei Xu, Roy Chris Willis, Angela Burrell, Mangkey Bounpheng, Xingwang Fang • Ambion, Inc.
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