Product details

Anewex RNA cleanup kits provide a simple, and quick way to clean up your RNA from pre-purified RNA (by phenol/ chloroform), enzymatic reactions (amplification or labeling reactions), DNAse digestions, etc.

• Quick procedure delivering high-quality total RNA in minutes
• Ready-to-use RNA for high performance in any downstream application
• No phenol/ chloroform extraction, no CsCl gradients, no LiCl or ethanol precipitation
• Complete removal of RT-PCR inhibitor

 

Applications

RNA isolated with Anewex technology has A260/280 ratios of 1.9-2.1, and A260/230 and are applicable for any downstream assays

• RNA-seq
• Quantitative, real-time RT-PCR
• End-point RT-PCR
• Northern, dot, and slot blotting
• Array analysis
• Poly A + RNA selection

 

Protocol

1. Add 2 volume (recommend: 100 µL) of RCB into 1 volume (recommend: 50 µL) of RNA in 1.5 mL tube.
2. Mix well, then transfer the sample to RNA cleanup spin column placed in a 2 mL collection tube.
3. Close the lid gently and centrifuge for 15 s at ≥10,000 rpm. Discard the flow-through
4. Add 300 µL RCW1 to the spin column.
5. Close the lid gently and centrifuge for 15 s at ≥10,000 rpm. Discard the flow-through
6. Add 500 µL RCW2 to the spin column.
7. Close the lid gently and centrifuge for 15s at ≥10,000 rpm. Discard the flow-through
8. Place the RNA cleanup spin column in a new 1.5 mL collection tube. Add 15 µL  RNase-free water directly to the center of the spin column membrane.
9. Close the lid gently and centrifuge for 1 min at full speed to elute RNA. 

Do not elute with less than 10 µL RNase-free water as the spin column membrane will not be sufficiently hydrated.

 

Supporting data and figures

The efficiency of application such as real time RT-PCR and RNA-Seq depends strongly on the purity of the RNA sample used. To assess RNA purity, the absorbance of RNA at 260 nm and the absorbance of potential contaminants at 280 nm or 230 nm was determined by spectroscopic measurement (e.g. Nanodrop). An A₂₆₀/A₂₈₀ ratio of 1.8-2.1 at pH 7.5 is widely accepted as indicative of highly pure RNA. Pure RNA should also yield an A₂₆₀/A₂₃₀ ratio of around 2 or slightly higher. After using Anewex RNA cleanup kit for extracted RNA, an A₂₈₀/A₂₆₀ and A₂₃₀/A₂₆₀ was significantly improved from 1.7 to 1.96 for A₂₈₀/A₂₆₀ and from 0.65 to 2.17 for A₂₈₀/A₂₆₀.

 

Fig. 1: Quality of RNA, which was extracted from high fat diet liver tissue using guanidine thiocyanate /phenol/ chloroform, was measured by NanoDrop

 

Fig. 2: Quality of RNA, which was cleaned up using Anewex RNA cleanup kit, was measured by NanoDrop