Phenol-Free Total RNA Purification Kit

Phenol-Free Total RNA Purification Kit
  • Save time
    • Rapid and simple spin-column purification and concentration
    • Total RNA purification from multiple samples in 20 minutes
  • Reduce hazardous chemical use
    • Eliminate phenol and chloroform
    • Reduce interference by residual phenol in downstream applications including PCR
  • Enhance recovery of total RNA including microRNA species
    • Efficient recovery from small sample sizes
    • Applicable to a wide range of sources

AMRESCO's  Phenol-Free Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA.  In as little as 20 minutes, all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA), can be extracted without the use of phenol or chloroform. The kit is an excellent tool for studies comparing expression of miRNA to other RNA species since both miRNA and mRNA can be analyzed from the same sample without further purification. The kit can be used with samples from a wide range of organisms.

Enrichment of Small RNA Species

Phenol-Free Total RNA Purification Kit
Figure 1. Size Range of RNA purified with Phenol-Free Total RNA Purification Kit versus a competitors kit. Total RNA was isolated from 1 x 109 E. coli cells with Phenol-Free Total RNA Purification Kit and a competitor’s kit. Five µL and 1 µL isolated RNA was analyzed on an agarose gel (Panel A) and the Agilent® 2100 BioAnalyzer RNA Nano 6000 chip (Panel B), respectively. Note the presence of small RNA species (red circle) in the samples isolated via Phenol-Free Total RNA Purification Kit and the absence of these RNA species in the competitor RNA preparation.

Amplification of miRNA and mRNA from the Same Extraction

Amplification of RNA purified with Phenol-Free Total RNA Purification Kit
Figure 2.  Amplification of total RNA isolated from Phenol-Free Total RNA Purification Kit versus competitor kits. Blue: Amplification of RNA from Phenol-Free Total RNA Purification Kit; Red: Amplification of RNA from a competitor's phenol-based extraction reagent; Green: Amplification from competitor's spin-column technology.  RNA isolated from 0.75 million HeLa cells was polyadenylated according to Shi and Chiang (2005). Four microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was used in a 20 µL qPCR reaction with primers to the human microRNAs (miR-19 and miR-21) and large mRNA (S15). Amplification of RNA purified with Phenol-Free Total RNA Purification Kit matched amplification of both miRNAs and mRNA amplified with the phenol based extraction reagent. Amplification of microRNAs from the competitors spin column technology was not detectable.

Purification of Total RNA from Different Sources and Tissue Types

Phenol-Free Total RNA Purification Kit
Figure 3. Total RNA was isolated from 5 x 108 E. coli cells, 1 x 106 HeLa cells, 100 µL rat blood and 10 mg hamster kidney. One microliter of the isolated RNA was analyzed on the Agilent® 2100 BioAnalyzer using an RNA Nano 6000 chip. RNA from a variety of sources extracted with Phenol-Free Total RNA Purification Kit consistently scored a RIN value between 8 and 10.

Sensitivity of Phenol-Free Total RNA Purification Kit

Phenol-Free Total RNA Purification Kit Sensitivity
Figure 4. RNA Recovery by Phenol-Free Total RNA Purification Kit Approaches Single Cell Levels. Total RNA was extracted from serial dilutions of 293 HEK cells over a range of 1x106 cells to a single cell. The RNA was polyadenylated and reverse transcribed with an oligo dT primer. Three µL of the reverse transcription reaction was amplified by PCR  with primers to the human beta-actin gene. PCR products of beta-actin were detected from as little as a single cell. M is the marker lane.

Consistent Isolation of Large and Small RNA Species as a Function of Starting Amounts

Phenol-Free Total RNA Purification Kit
Figure 5. Total RNA was isolated from 10 to 100,000 HeLa cells with Phenol-Free Total RNA Purification Kit (blue), a competitor’s spin-column kit (green) and a phenol-based RNA extraction method (red). Relative expression of miR-21 (Panel A), and S15 (Panel B) was determined by RT-qPCR of total RNA samples. One microliter of isolated RNA was  subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Two microliters of the reverse transcription was added to a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15 transcripts (Panel B). The resulting threshold cycle (Ct) values were plotted against input cell number. RNA isolated with Phenol-Free Total RNA Purification Kit had the best linearity (higher R2) and sensitivity (lower Ct) for both large RNA (S15) and small RNA (miR-21).

Consistent Detection of miRNA from Plasma

Phenol-Free Total RNA Purification Kit
Figure 6. Isolation of Total RNA from Plasma by Phenol-Free Total RNA Purification Kit versus Competitors. Total RNA was isolated from 50, 100 or 200 µL of rat plasma in triplicates using Phenol-Free Total RNA Purification Kit (blue), a competitor spin column kit (green) and a phenol-based RNA extraction method (red). Two microliters of the  isolated RNA was subjected to a 20 µL reverse transcription with miR-21 stem-loop reverse primer or oligo dT primer. Three microliters of the reverse transcription reaction was transferred to a 20 µL real-time PCR reaction with primers to the human miR-21. Phenol-Free Total RNA Purification Kit is the only product that showed (1) consistent detection of miR-21 transcripts across all input volumes and (2) Ct values correlated to input volume (decrease Ct with increase input).

Consistent Performance of Diverse miRNA from Plasma in Downstream Applications

Phenol-Free Total RNA Purification It
Figure 7.  Total RNA including miRNA was isolated from 100 µL of mouse plasma in duplicate with Phenol-Free Total RNA Purification Kit, Competitor A spin-column Kit or a phenol-based RNA extraction method. One hundred nanograms of extracted RNA from each kit was applied to an Illumina microRNA expression profiling kit. Scatter plots display better consistency (better clustering) of replicate signals from AMRESCO 's samples. Genes with Pval < 0.01 for both replicates were in blue. The associated table suggested that AMRESCO's protocol recovered the same diversity of miRNA with better consistency (higher r2 value).

Enhanced Diversity of miRNA from Plasma

Phenol-Free Total RNA Purification Kit
Figure 8: miRNA from plasma isolated with Phenol-Free Total RNA Purification Kit exhibits greater diversity than a leading competitor. Total RNA was isolated from 100 µL of plasma using AMRESCO's Phenol-Free Total RNA Purification Kit or 625 µL of plasma using Competitor A's miRNA Kit and was applied to an NCode expression profiling kit. Microarray images suggested that Phenol-Free Total RNA Purification Kit (left) achieves greater diversity of miRNA from smaller input amount of plasma than the competitor’s miRNA kit (right). Image courtesy of LC Sciences, Houston (www.lcsciences.com).

Kit Specifications

 Binding Capacity per Column
     Up to 50 µg RNA
 Maximum Loading Volume Per Spin Column   
    650 µL
 Size of Purified RNA
    All sizes, including < 200 nt     
 Time to Complete 10 Purifications          
       20 minutes
 RNA Yields
  
      Tissue  
           Liver (10 mg)
       30 µg
           Kidney (10 mg)       10 µg
           Brain(10 mg)       12 µg
           Blood (hamster, 100 µL )       5 µg
      Cells  
           Hela (1.0 x 106)       15 µg
           CHO (1.0 x 106)       11 µg
           Yeast (1.0 x 106)       30 µg
           E. Coli (1.0 x 106)       43 µg

Kit Components

1. Spin Columns
2. Lysis Solution
3. Wash Solution
4. Elution Buffer
5. Collection Tubes
6. Product Insert

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   AMRESCO News  

  • AMRESCO LLC featured in Genetics Engineering & Biotechnology News October 2011. Download Article.
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