Gel image: RT-PCR of B-actin (240bp) from amplified RNA. Table: Amplified RNA was quantified on NanoDrop spectrophotometer.
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The aim of this kit is to amplify total RNA when you have limited amounts of starting material. In our case, we were looking for tumor cells which were present in patients’ blood . After isolating those tumor cells from blood, we isolated total RNA from them. The number of tumor cells varied from every patient. Once we got the total RNA (from limited cells), we used this kit to amplify RNA in order to get micrograms of RNA which was then used in many applications.
Amplified RNA was converted to cDNA. cDNA was then used for RT-PCR and qRT-PCR.
<1 ng of total RNA obtained from a limited number of tumor cells.
This is a two round amplification process . 1. First-strand cDNA Synthesis. Poly(A) RNA of a total RNA sample is reverse transcribed into first strand cDNA. The reaction is primed from an oligo(dT) primer containing a phage T7 RNA Polymerase promoter sequence at its 5'-end. First strand cDNA synthesis is catalyzed by SuperScript™ III Reverse Transcriptase (from Life Technologies, provided by the user) and performed at an elevated temperature to reduce RNA secondary structure. 2. Second-strand cDNA Synthesis. The RNA of the cDNA:RNA hybrid produced in Step 1 is digested into small RNA fragments using an RNase H enzyme. The RNA fragments then prime 2nd strand cDNA synthesis. The resulting double-stranded cDNA contains a T7 transcription promoter in an orientation that will generate anti-sense RNA (aRNA; also called cRNA) during the in vitro transcription reaction. The cDNA produced can be used in the in vitro transcription reaction without the need for purification. 3. In Vitro Transcription of aRNA or Aminoallyl-aRNA. High yields of aRNA, aminoallyl-aRNA, or biotin-aRNA are produced in a rapid in vitro transcription reaction that utilizes the double-stranded cDNA produced in Step 2. 4. Purification of the aRNA (aminoallyl-aRNA) The aRNA, aminoallyl-aRNA, or biotin-aRNA produced in Step 3 is purified by spin column chromatography (from Life Technologies, supplied by the user). The TargetAmp 1-round amplification procedure is now complete. The TargetAmp 2-round amplification procedure continues with steps 5-8. 5. Round Two, 1st-Strand cDNA Synthesis. The aRNA produced and purified in the first round amplification process is reverse transcribed into first strand cDNA using SuperScript II Reverse Transcriptase (Invitrogen; supplied by the user). The reaction is primed using random sequence hexamer primers. 6. Round Two, 2nd-Strand cDNA Synthesis. The RNA of the cDNA:aRNA hybrid produced in Step 5 is digested to small RNA fragments by RNase H. Second-strand cDNA synthesis is then primed using a T7-Oligo(dT) Primer. The resulting product is a double-stranded cDNA containing a T7 transcription promoter in an orientation that that will generate aRNA during the second round in vitro transcription reaction. 7. In Vitro Transcription of aRNA (AminoallylaRNA). High yields of aRNA (or aminoallyl-aRNA) are produced in an in vitro transcription reaction that utilizes the double-stranded cDNA produced in Step 6. 8. Purification of the aRNA (aminoallyl-aRNA) The aRNA (aminoallyl-aRNA) produced in Step 7 is purified by spin column chromatography (supplied by the user). The TargetAmp 2-Round amplification procedure is now complete.
Read and follow the manual very carefully. Before performing a crucial experiment, please gather everything, then start.
In my case, I had very limited numbers of cells ~ 100-500; I had a few cells from one patient sample and little more from another patient sample. But the end results were very similar; I ended up getting ~20-35 micrograms of total RNA. I am very happy with the yield of amplified RNA and the quality was also very good. I quantified and qualified these RNA samples with a NanoDrop spectrophotometer and denaturing gels. (See figure for typical results.) I end up getting good quantity and very good quality of amplified samples.
The protocol is very lengthy, but self-explanatory, and the end result is good.
Product Name: TargetAmp™ 2-Round aRNA Amplification Kit 2.0Supplier Page