ArrayPure™ Nano-scale RNA Purification Kit

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Last modified: 2008-07-15 14:57:15

The ArrayPure™ Nano-scale RNA Purification Kit provides all of the reagents needed to purify RNA from one to a few thousand eukaryotic cells, such as obtained from Laser Capture Microdissection (LCM) procedures. The reagents are all aqueous to avoid the use of toxic organic solvents.

This nano-scale protocol has been developed and tested with quantitative real-time PCR on 1 to 10,000 eukaryotic cells (see Figures). Cell numbers were based on living cells that were enumerated and then diluted.

The average yields of RNA from 1,000 and 10,000 HeLa cells, as determined by RiboGreen® fluorescence, were 21 and 213 ng, respectively. In addition, µg amounts of RNA have been produced from 20 HeLa cells using aRNA 2-round synthesis techniques.

Figure 1. Quantitative Real-Time PCR Extension Profiles Intact HeLa cells were serially diluted ten-fold in growth medium to dispense 104, 103, 100, 10 or 0 (medium control) cells per tube. (Note: these were actual dilutions of living cells and not dilutions of a cell lysate, as some commercial vendors have done.) The RNA in each tube was purified with the ArrayPure Nano-scale RNA Purification Kit. The purified HeLa RNA was converted to cDNA using EPICENTRE MMLV reverse transcriptase. These corresponding cDNAs were amplified by the quantitative FailSafe™ Real-Time PCR System and SYBR® Green I on a Bio-Rad iCycler iQ™ Real-Time PCR Detection System. The cycling conditions were 95°C for 2 min and then 45 cycles of 95°C for 20 sec, 53°C for 30 sec, and 72°C for 30 sec. The primers used were for human cyclophilin A (peptidylprolyl isomerase A) 5'- CAT ACG GGT CCT GGC ATC TTG and 5'- GCC ATT CCT GGA CCC AAA GC.

Figure 2. Linear Plot of CT vs Log of Initial HeLa Cell Number The log of initial cell number for the amplified cDNA made from the RNA purified from the 10, 100, 103 and 104 HeLa cells was plotted against cycle threshold (CT) values. Slope: -3.75; r2 = 0.991. The CT linearity extends for 4 orders of magnitude of initial cell number.

Figure 3. Melt Curve Analysis of the Quantitative Real-Time PCR. The cDNA corresponding to RNA purified from 10, 100, 103 and 104 HeLa cells yielded PCR products (peaks at 87°C to 88°C). The 0 cell (medium control; black line) sample yielded only primer dimers (peak at 80°C to 81°C) indicating the absence of detectable RNA, as expected.

Figure 4. Quantitative Real-Time PCR Amplification Plot. HeLa cells were grown in tissue culture, aseptically diluted and trapped inside sterile 5µl microcapillary pipets. The number of cells isolated was verified by observation with an inverted microscope. Then, the cell(s) was/were eluted by centrifugation from the capillary pipet, washed with phosphate buffered saline, and the RNA purified by ArrayPure™ Nanoscale RNA Purification Kit. Purified HeLa RNA was converted to cDNA using EPICENTRE's MMLV reverse transcriptase. The corresponding cDNAs were amplified using FailSafe™ PROBES Real-Time PCR PreMix-Choice Kit (PreMix 3). Cycling conditions were 94°C for 2 min and then 35 cycles of 95°C for 10 sec, 60°C for 10 sec, and 72°C for 30 sec. The primers used were for human beta-actin 5'- TGG ACA TCC GCA AAG ACC TG and 5'- CCG ATC CAC ACG GAG TAC TT. The TaqMan Probe was 5'- TET-CAC CAC CAT GTA CCC TGG CAT TGC C-BHQ1. Real-Time PCR results using a Bio-Rad iCycler iQ™ Real-Time PCR instrument are shown in triplicate for RNA from an average of 100, 10, 1 and 0 (medium alone) cells.

Figure 5. CT vs Log of Initial HeLa Cell Number is Linear. The log of the initial cell number for the amplified cDNA made from the RNA purified from an average of 100, 10 , and 1 HeLa cell was plotted against cycle threshold (CT) values. Slope = -3.14; r2 = 0.992. The CT linearity extends for 3 orders of magnitude of initial cell number.

Figure 6. HeLa cell RNA was purified from two separate tissue culture flasks, labeled with Cy™3 or Cy™5, and hybridized to a microarray containing Operon's Array-Ready Human Oligo (70-mer) Set™.

Last modified: 2008-05-14 16:46:46

Applications

• RT-PCR
• aRNA
• LCM

Benefits

• Quantitatively isolate RNA from as few as 10 cells
• Purify RNA for aRNA synthesis
• Simple, nontoxic procedure

Last modified: 2008-05-14 16:46:46

Protocols for: ArrayPure™

(catalogue number MPS04050)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. Emmert-Buck, M.R. et al., (1996) Science 274, 998.
2. Bonner, R.F. et al., (1997) Science 278, 1481.
3. Miller, S.A. et al., (1988) Nucl. Acids Res. 16, 1215.

Last modified: 2008-05-14 16:46:46