Superscript Vilo cDNA synthesis kit (qPCR step 4)

The SuperScript® VILO™ cDNA Synthesis Kit is designed to increase cDNA yields and improve the dynamic range of your qRT-PCR assays. This means that you obtain the same relative representation in your cDNA, irrespective of the abundance level of your gene of interest. Looking for consistent results for your RT reactions? Unprecedented linearity across the broadest range of input material? You'll get them with the SuperScript® VILO™ cDNA Synthesis Kit: For probe-based two-step qRT-PCR, linear cDNA yields from 1pg to 2.5ug of total RNA in 20ul reactions. For SYBR® GreenER™ Two-Step qRT-PCR, linear cDNA yields from 1pg to 100ng of total RNA in 20ul reactions. Reaction volumes can be scaled up to 100ul if more cDNA is needed, while maintaining the same linearity of the reaction.

Excellent linearity in your RT step means you will: obtain the same relative representation in your cDNA qPCR template, regardless of gene abundance during normalization, get both your gene of interest and the reference gene in a linear phase for cDNA synthesis even if their abundances differ in the starting material get greater accuracy in your qRT-PCR data by reducing this common bias.

The other videos in this series are:
Introduction to the steps of the qPCR workflow (qPCR step 1):
   • Introduction to the steps of the qPCR work...  
Isolate RNA with the PureLink™ RNA Mini Kit (qPCR step 2):    • Isolate RNA with the PureLink™ RNA Mini Ki...  
Quantitate RNA with the Qubit® 2.0 Fluorometer (qPCR step 3):
   • Quantitate RNA with the Qubit® 2.0 Fluorom...  

Fast SYBR Green vs. TaqMan® Fast Advanced Master Mix (qPCR step 5):
   • Fast SYBR Green vs. TaqMan® Fast Advanced ...  
Amplify Sample with The StepOnePlus™ Real Time System (qPCR step 6):
   • Amplify Sample with The StepOnePlus™ Real ...  
Preparing the card for the Viia™ 7 with TaqMan® Assays (qPCR step 7):
   • Preparing the card for the Viia™ 7 with Ta...  
Amplify sample with the Viia™ 7 Real-Time PCR system (qPCR step 8):
   • Amplify sample with the Viia™ 7 Real-Time ...  

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Transcript:

Let's make some CDNA. Now we've isolated our RNA. We qubited it, and we know what its quantity is. Let's make CDNA. In order to do this, we are going to use the Super Script Vilo CDNA synthesis skit. Let's open it up.
First of all, we have our nice little insert that tells us everything we need to know about the kit. And now, there are just 2 tubes in here. One is the 5X Vilo reaction mix. And 2 is the 10X Superscript enzyme mix. I'll put both of these on ice. Let's talk a little bit about this kit.

And the 2 components. It includes Superscripts, 3RT, the RNAsout, recombinant ribonucleus inhibitor, and a proprietary helper protein.

And the 5x Vilo reaction mix contains random primers, magnesium chloride, and DNTPs in a buffer formulation that has been optimized for QPCR or real time PCR.

Now what's really nice about this kit, is that it is going to maintain linearity across a wide range of RNA inputs. So, you can do as much as 2.5 micro liters of RNA and convert that into cDNA in just a 20 micro liter reaction. It's pretty amazing. In addition, you will maintain linearity when you assay with QPCR.

Here's how it goes. We are going to need 4 components. Your 10X Superscript mix, your 5X Vilo Mix, which are both provided in this kit.
Some water that's RNAse free.

And your RNA sample. That's it. Mix these four together then you will end up with converting RNA into CDNA ready to go into real time PCR.

For more information, please visit: http://www.invitrogen.com/site/us/en/...