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AnyGenes

FREQUENTLY ASKED QUESTIONS

frequently asked questions : question: list 1Sample preparation
frequently asked questions : question: question 1Which tests do you advise to assess the quality of RNA samples ?
In order to perform analysis with our SignArrays® and ensure high quality results, all RNAs must be assessed by spectrophotometry to measure the relative amount of RNA in each sample and determine their quality using the following criteria: The total RNA concentration must be greater than 40 µg/ml The A260/A280 ratio should be between 1.8 and 2.0 The A260/A230 ratio should be greater than 1.7 Moreover, electrophoresis on agarose gel allows to check the integrity of RNA.
frequently asked questions : question: question 2How can we send you samples ?
You can send us your samples as: dried cell pellets or with Trizol (about 1ml for 1 to 10 x 106 cells or up to 100 mg of tissue) solutions of high quality RNA diluted with “RNAse-free” water. We recommend you sending us your samples in a polystyrene container filled with dry ice. The tubes must be clearly annotated, identified and accompanied by a descriptive document with all the required data for the analysis. You will receive a detailed sending protocol with your quote.
frequently asked questions : question: question 3What is the required amount of cDNA for a SignArray analysis ?
The recommended cDNA amount for an analysis with a 96-well plate SignArray corresponds to almost all of the RT product made from an amount of between 500 ng and 1 μg of total RNA. For a 384-well plate, twice more cDNA is required. However, lower amounts of RNA can also be used.
frequently asked questions : question 4 Should we perform our analysis in duplicate or triplicate ?
In order to overcome eventual experimental variations during the upstream steps of the qPCR array (cell culture, RT,…), it is recommended to perform your experiment in triplicates from the beginning of the protocol. However, for the same cDNA, perform qPCR arrays in triplicate is not required since this technology is reliable, robust and reproducibl
frequently asked questions : question: list 2qPCR and SignArrays®
frequently asked questions : question: question 5How AnyGenes® controls the quality of the primers ?
In order to perform analysis with our SignArrays® and ensure high quality results, alEach pair of primers was validated in our laboratory. Thanks to very stringent design methods, these primers allow a specific, highly sensitive and reproducible amplification. In fact, our feature is based on the design of our primers, which is carried out on two exons, thereby avoiding amplification of genomic DNA contamination. Upon receipt, a quality control is performed for all pairs of primers and should therefore ensure reproducible results, qPCR optimal efficiency and a lack of primer-dimers formation. The specificity of the primers to their target cDNA sequence is also validated through the presence of a single peak at the melting curves and a single band on agarose gel corresponding to the expected size of the amplicon.
frequently asked questions : question: question 6What should I do if my sample is contaminated with genomic DNA ?
A good qPCR laboratory practices must be respected to avoid any contamination, ie handle in a space called "DNA-free". Respect the extraction protocol recommended by the supplier. If traces of genomic DNA exist, we advise you to add a DNAse treatment step after RNA extraction. Moreover, our design minimizes the genomic DNA quantification.
frequently asked questions : question: question 7What are the quality controls in your SignArrays ?
The SignArrays plates have four quality controls: 2 negative controls to ensure the quality of your preparation reaction mix 2 positive controls to check the qPCR performance. Moreover, a very low value of the positive controls standard deviation allows to check the reproducibility of qPCR reaction with our kit and your samples.
frequently asked questions : question: question 8Which type of qPCR instrument should I use with your SignArrays ?
Our SignArrays are compatible with most of qPCR instruments. We will supply the adapted SignArrays plate format, mentioned during your order.
frequently asked questions : question: question 9How much does a SignArrays plate cost ? And an analysis in your laboratory ?
The price of our SignArrays depends on the amount of ordered plates and the number of tests you want to perform. We offer very attractive prices and the most competitive of the market.
frequently asked questions : question: question 10What would happen if I used my own mix with SignArrays ?
We recommend using our SignArrays with our reagents that we have optimized for this use. However, you can use reagents provided from others suppliers of your choice, but we could not guarantee the obtained results, as we did not tested them with our SignArrays.
frequently asked questions : question: question 11What types of fluorescent markers do you use in your SignArrays ? Do you use probes ?
All plates SignArrays standards are used with SYBR® Green. However, we are offer SignArrays with specific probes.
frequently asked questions : question: question 12How long it takes to perform an analysis ?
For already designed signaling pathways, it will take 1-2 weeks. For personalized SignArrays, it will take 4-5 weeks. Contact us for large quantity of samples.
question 13What is the amplicons size ?
Primers are specifically designated to generate amplicons between 70 and 90 bps. This design is also adapted for FFPE analysis.
question 14Your products have been referenced in publications?
Our reagents Perfect MasterMix SYBR® Green and Perfect MasterMix Probe have already been referenced in several publications. Several publications are in progress for our SignArrays.
What is the efficiency of qPCR with SignArrays ?
Oligos present in our SignArrays enable a qPCR efficiency between 95 and 105% with our Perfect MasterMix.
list 3Data Analysis
question 19How can I get the results ?
The results report will be sent to you in PDF and Excel format.
question 15Which analysis method do you use ?
After normalization with the selected house-keeping genes, the analysis method used by AnyGenes® is based on the ΔΔCt calculation, for relative quantification of transcripts.
question 16My CT values are very high (CT>35). Why ?
Several parameters can lead to high values ​​of CT: The starting amount of RNA is very low, we advise you to increase the starting amount of RNA The quality of extracted RNA are of poor quality, we strongly recommend to check the protocols and perform the required quality controls. Some troubles occurred during the RT step (Reverse Transcription step), we also recommend to check the protocols and perform the required quality controls. Genes of interest are not expressed in your samples
question 17How can I select the House-Keeping Genes (HKG) for the normalization step ?
It is well known that the expression of HKG can vary from one cell type to another. For this reason, we include in our SignArrays 8 HKG in order to give you more choice to select the most stable and the most appropriate HKG for your study. We also advise you to use specific softwares like geNorm, Normfinder, GenEx... to select the appropriate HKG.
list 4Technical support
question 18How can I get technical support for your SignArrays products ?
You can call at 01-45-17-13-54 for technical questions or also send an e-mail to technical@anygenes.com. Our offices are open from Monday to Friday from 9:30 to 19:00.
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