StellARray™ gene expression (cDNA): A single run on a 384-well plate requires the product of a cDNA synthesis reaction acting on 50-250 ng RNA.
StellARray™ copy number variation (genomic DNA, gDNA): A single run on a 96-well plate requires 125 ng gDNA, a 384-well plate requires 500ng gDNA.

Rigorous testing and strong selection facilitated the development of XenoQ™. XenoQ™ gene expression and copy number assays exclusively amplify xenograft-derived sequences even when xenograft-recipient-sequence accounts for 94% of the total template material.

XenoQ™ gene expression (cDNA): A single run on a 384-well plate requires the product of a cDNA synthesis reaction acting on =125 ng xenograft-derived RNA
XenoQ™ copy number variation (gDNA): A single run on a 384-well plate requires =60ng xenograft-derived gDNA.

If a qPCR run is interrupted (such as by a power cut/outage), the plate must be discarded and the run repeated on a new plate. BHB does not expect you to cover the cost of such extra work and materials. However, our expected turnaround time of two weeks depends on the ability to repeat qPCR runs, so BHB strongly suggests sending at least twice as much DNA per sample as is required by a single run.

cDNA samples can be rendered useless by RNase activity or the presence of gDNA. Recommendations for sample prep can be found below.
Prior to synthesizing cDNA, all RNA preparations should have:
        1. 260nm:280nm UV-vis spec ratio of 2*
        2. Electropherograms with no signs of RNA degradation or genomic DNA contamination**

gDNA samples (for Copy Number Variation, CNV studies) should have these qualities:
        1. PCR-amplifiable
        2. 260nm:280nm UV-vis spec ratio of 1.8*

        * Determined via NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.).
        ** Determined via Agilent® BioAnalyzer® 2100 or Bio-Rad® Experion™; please
          refer to Assessing the Quality of RNA.

Samples for StellARray™ gene expression analysis should contain the cDNA synthesis reaction mixture (20 µl) diluted with 180 µl of nuclease-free dH₂O.
For XenoQ™ gene expression studies please refer to the XenoQ™ Brochure.
Samples for StellARray™ and XenoQ™ CNV studies should contain between 10-100ng gDNA/µl.

For optimal statistical results
We recommend using at least 3 replicates per experimental group. Gene Expression studies should employ "biological replicates" (refer to our GPR page for more information). Copy Number Variation (CNV) studies utilize "technical replicates" and/or "bio-tech replicates".

Technical replicates perform the same test multiple times on one DNA preparation. Variation among results from technical replicates should represent variation in the testing method.

Bio-tech replicates involve collecting multiple samples of tissue from a single organism, processing these samples in parallel, and then performing the same test on each. Variation among results from bio-tech replicates should indicate variation in the source organism.

The method employed for extraction/preparation of nucleic acids should be the same among all your experimental groups.

BHB recognizes that each user may have a preferred RNA purification method and/or cDNA synthesis method. It is vitally important to prepare the most pristine RNA possible. The recommendations below are simply a means to communicate our own experience and may not entirely reflect the exact protocol necessary for your specific samples.

It is important that the sample collection and sample prep process be as pristine as possible to generate the most robust quantitative PCR data. Through years of experience, BHB has established minimum requirements for biological sample collection and storage along with the purification, quantification, and qualification of nucleic acid template to be used in the generation of quantitative PCR data. Following these guidelines the user should experience a higher level of success.

• Use DNase/RNase - free plastics (not autoclaved).
• Handle tubes with forceps treated with RNaseZap® (Cat# 9780 or 9786, Life Technologies, Inc.).
• Clean gloved hands and surfaces with RNaseZap®.

RNA
It is advisable to collect the biological sample as rapidly as possible, minimizing the time between the collection and the stabilization step. If RNA is purified from tissue it should be rapidly stabilized in RNAlater® (Cat# 7021, Life Technologies, Inc.) following the manufacturer's recommendations. Alternatively, the tissue can be placed directly into lysis buffer and immediately homogenized. Rapid processing can insure the purification of high quality RNA.

BHB has successfully purified high quality RNA using the RNAqueous®-4PCR kit (Cat# 1914, Life Technologies, Inc.) following the manufacturer's recommendations.

It is very important to recognize that small amounts of tissue provide higher quality RNA and better yields. Do not increase the size of the tissue beyond the recommended amounts as this will not generate higher yields of high quality RNA. On the contrary, it will likely lead to degradation of the RNA and possibly clog the membrane. By using small sample sizes it is likely that the sample will be completely homogenized thus decreasing residual tissue pieces that will be captured on the membrane. These tissue pieces carry RNase that will likely be eluted along with the RNA and thereby degrade it. To obtain a small sample (sample size is defined in the RNAqueous®-4PCR kit protocol booklet) we either excise the appropriate sized sample directly from the source or from a RNAlater®-stabilized sample.

To process a sample from an RNAlater®-stabilized tissue sample we recommend first preparing an RNase-free work surface by placing a sheet of Parafilm® M (Cat# 01851-AB, SPI supplies) onto the bench and wipe it with a RNaseZap® wipe followed by 95% ethanol. The tissue sample is removed from the RNAlater® onto the Parafilm® and the desired amount of tissue is excised using an RNase-free surgical blade (prepared by wiping with RNaseZap® followed by 95% ethanol). The original piece of tissue is rapidly returned to RNAlater® and the sample is placed into lysis buffer for further processing. We routinely use a RNase-free pipette tip for tissue handling.

Tissue Homogenization
Process small solid tissue pieces in the recommended volume of lysis buffer (350 µ;l) contained in a 1.5 ml Low-adhesion micro centrifuge tube (Cat# 1415-2600, USA Scientific). The lysis buffer is added first to the tube followed by the tissue piece and the tissue is immediately but partially homogenized by hand pressure and rotation using a disposable plastic pestle (Cat# 1415-5390, USA Scientific). Then a cordless motor is used to rapidly spin the pestle for complete homogenization of the tissue. Alternatively, we have also successfully employed a bead-beater system such as the FastPrep system (MP Biomedicals). For fibrous tissues we have used a combination of the FastPrep system with the Fibrous Tissue RNA prep kit (Cat# 74704, Qiagen).

RNA Elution and Treatment
To increase the likelihood of obtaining and maintaining high quality RNA it is recommended that SUPERase⋅In™ (a RNase-inhibitor, Cat# 2696, Life Technologies, Inc.) be added to the elution tube prior to the elution of the RNA. SUPERase⋅In™ is effective at 1 U/µL, it is normally supplied at a concentration of 20 U/µL. Make sure to add enough SUPERase⋅In™ so that is at or above its effective concentration in the final (total) elution volume. After the first elution volume is collected the solution should be mixed by tapping followed by the second elution (as described for the RNAqueous®4-PCR kit).

DNA is effectively eliminated from the RNA prep using the DNA-free™ system (comes as part of the RNAqueous®4-PCR kit) (individually: DNA-free™, cat# AM1906; Life Technologies, Inc.) and it MUST NOT be skipped. DNA is present in all RNA preps prior to DNase treatment and will contribute erroneous signal during SYBR Green-based quantitative PCR (and even possibly probe-based detection systems). The DNA-free™ kit utilizes RNase-free rDNase I, 10X buffer, and Stop slurry (or DNase Inactivation Reagent). We routinely use this system with slight modification; we use 2 µL rDNase I instead of 1 µL and we incubate for 1 hour at 37°C instead of 30 minutes. The Stop slurry binds and inactivates proteins. This removes rDNase I and SUPERase⋅In™ from the RNA solution. Subsequent it the DNase inactivation step, it is essential to centrifuge the sample at 10,000 x g for 1.5 minutes and, without disturbing the Stop slurry pellet, transfer the RNA solution to a new tube containing a replenishing amount of SUPERase⋅In™. Again, enough SUPERase⋅In™ should be added so that it is at a concentration of at least 1 U/µL. Assume that all SUPERase⋅In™ added during the elution step was removed or inactivated by the DNase I treatment.

Remove an aliquot (5-7 ul) of the DNase-treated RNA for quantification and qualification. Using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.) will provide RNA concentration data without much loss of sample (typically 1.5-2 ul). This quantity helps to determine the appropriate analysis mode for the Agilent BioAnalyzer 2100/Bio-Rad Experion, i.e. which chip should be used (the Nano or the Pico chip for the BioAnalyzer/the RNA HighSens chip or RNA StdSens chip for the Experion). These machines provide quantity and quality data. You must evaluate the data carefully, paying special attention to electropherograms (link to 'Assessing the quality of RNA.docx').

RNA purified in this manner can be stored at -80°C indefinitely. However, the RNA must be evaluated by the use of the Agilent BioAnalyzer 2100 or Bio-Rad Experion prior to freezing and after thawing.

cDNA Synthesis
We have had great success using the MessageSensor™ RT Kit (Cat#1745, Life Technologies, Inc.) with either random decamers (Cat#5722G, Life Technologies, Inc.) or an equal mix of random decamers and oligo dT primers (Cat#5730G, Life Technologies, Inc.). We do not recommend the use of gene-specific primers as the cDNA will be distributed to multiple wells containing a unique primer set.

We recommend using 100 ng - 500 ng of total RNA per 20 ul reaction (although I have had success with as little as 25ng total RNA). When using multiple samples it is best to prepare a master mix of all of the reagents (except the RNA) sufficient for the number of samples being processed plus a 10% excess to compensate for pipetting errors during the aliquotting of the master mix.

The MessageSensor™ RT kit requires that the RNA + H2O volume does not exceed 10 ul as the master mix added is 10 ul and the final volume of the reaction is 20 ul. To do this efficiently, use a 96-well PCR plate (handled in a RNase-free manner), prechilled using a cold block (avoid ice as it carries RNases and often contaminates samples).

The best case scenario is to 'equalize' your input RNA amount for the cDNA reaction. We do this using the quantities determined via a NanoDrop™ spectrophotometer (just be consistent with the quantification method, i.e. UV/Vis Spec or Agilent Bioanalyzer 2100 concentration values). Using the sample with the LOWEST concentration, calculate the total RNA mass in 10 ul of that sample. This mass will be the amount for all other samples, so determine the volumes to achieve this mass of RNA per sample and also determine the volume of nuclease-free dH2O (Cat# 9934, Life Technologies, Inc.) needed per sample to achieve a final volume of 10 ul. Add the appropriate volume of water for each sample to each well followed by the appropriate volume of RNA. Aliquot 10 ul of the master mix to each well and mix by pipetting but avoid bubbles. Also, ensure there are no bubbles at the bottom of the wells. Then seal the plate using a seal that is compatible with PCR but easily removable. We use the MicroSeal A Film (Cat# MSA-5001, Bio-Rad).

Incubate the plate in a thermal-cycler with a heated lid, programmed as follows: Hold at 25°C for 10 minutes, followed by
Hold at 42°C for 60 minutes, followed by
Hold at 95°C for 15 minutes, followed by
Hold at 4°C for infinity (or at least 10 minutes).

Remove the heated lid of the thermal-cycler (while the plate is at 4°C), let the plate cool down until it is comfortable to the touch, and then remove the plate from the cycler. Ensure that the solutions are not dislodged when carefully removing the plate (as it sometimes sticks in the block) and the reaction can splash onto the seal.

cDNA Sample Handling and Plate Set-up
We prefer to remove the cDNA's from the reaction well and store in 1.5 ml tubes. This ensures that there is no sample loss due to evaporation (PCR plate seals are notorious for poor cold-temperature sealing). To do this, remove the plate seal and add 180 ul of nuclease-free dH2O to each well for a 1:10 dilution. Mix each well by gently pipetting up and down and then remove the cDNA to a new, RNase/DNase-free 1.5 ml tube (catalog# 1415-2600, USA Scientific).

This test is performed pursuant to licensing arrangements with Roche Molecular Systems, Inc. and Applied Biosystems, LLC.