Frequently Asked Questions:
RNA or DNA Extractions:
Where can I get my samples extracted?
The VCC DNA Analysis Facility offers nucleic acid extractions as a service and can be ordered through your biodesktop account at https://biodesktop.uvm.edu/perl/desktop.cgi . The Microarray Facility staff can also assist investigators with the proper techniques and reagents when extracting RNA or DNA. Please email the staff or call 802-656-3936.
How do I properly prepare space for use with RNA extractions? When extracting RNA, the work space must be properly treated to eliminate the ubiquitous RNases. It is recommended to wipe down the area with a surface treatment agent such as RNase Zap (Life Technologies) or RNA Away (Molecular BioProducts). Whenever possible, perform all work in a PCR workstation. All plastics, glass, and consumables used in the process should be purchased as RNase-free or treated with RNase Zap, rinsed with Nuclease-free water, and autoclave or baked for 2hr. All reagents purchased should be RNase-free or made fresh with RNase free or DEP-C treated water.
Should I add an RNase Inhibitor to my RNA sample? It is always a safe precaution to add 10 units of RNase Inhibitor such as RiboLock from Fermentas corp (or equivalent), for every 30 ul volume of RNA. Likewise, RNase inhibitors may also be added to wash buffers or similar reagents that might come in contact with the cells or sample. When in question, don’t hesitate to contact the facility staff at http://vgn.uvm.edu/microarray/personnel.
What is the best method to use for extracting RNA from Cells? The Qiagen RNeasy Mini (>105 cells) or Micro kits (<105 cells) generally work well for eukaryotic suspended and adherent cells, but most Silica column-Guanidium lysis reagents are fine. For samples that are suspect of being high in extracellular biomolecules such as polysaccharide, adipose, or similar, a silica column may perform poorly and Trizol should be used.
What is the best method to use for extracting RNA from Tissue? A TriZol extraction using a high speed homogenizer such as the FastPrep system perfoms well. The use of an abrasive material such as diamond or garnet will assist with the homogenization. Further clean-up with a silica column is recommended.
What is the best method to use for extracting RNA from FACS? Please consult with the microarray staff for successful RNA recovery from cells sorted using flow cytometry. Several key points are highlighted below:
Preparing the flow cytometry is not a small task as it must be completely free of RNases from the sheath tank to the sorting nozzle. This decontamination procedure will take considerable time, so be prepared. Ensure the dip tubes, septa, flow cell, all tubing lines, and nozzles have been completely decontaminated with bleach, RNase ZAP, ethanol, autoclaving, or other qualifying technique prior to the sort. Rinse cytometer with DEPC water. Remember DEPC water DOES NOT inactivate RNases, and is only RNase-free water. The sheath fluid and tank must be RNase free. Use only sterile RNase free tubes on the cytometry that have never been open to contaminated air.
As a control, retain some cells and extract the RNA to determine the condition of RNA prior to the sort. Also before and after trypsinization for adherent cells. Often the flow cytometry, even after a good decontamination procedure can be the source of RNases. Whenever possible suspend cells in RNAlater prior to the sort and after trypsinization. Again, checking RNA integrity at this step is a good control. If RNAlater is not an option, you may consider adding RNase inhibitor to prevent degradation during a sort. Use RNase inhibitors that are DTT independent such as Ambion's Superase-In.
During the sort, sort cell directly into your extraction reagent, something like Trizol LS or Qiagen's RLT. When using TriZol, Only Use TriZol LS which is slightly more concentrated than regular regular TriZol. This formula allows lower quantities of reagent to be used relative to the amount of the sample. (Regular TriZol can tolerate 10 part TriZol to1 part sample volume while TriZol LS can allows 3:1. In the case of Guanidium Isothiocyanate (or Qiagens's RLT buffer) sort at a ratio of 100ul of sort liquid to 350 ul of RLT or a multiple of that.
Immediately after the sort, extract RNA according to the selected reagents manufacturer's protocol and evaluate the integrity of the RNA.
What is the best method to use for extracting RNA from LCM? Both the PicoPure and Qiagen Micro Kit perform well. With small recovery targets, it is recommended to skip the on-column DNase treatment to increase recovery of RNA as it is known that losses occur during this procedure. It is also recommended to elute off the column with water of 60 C and a slightly basic pH.
What is the best methodology to use for DNase treatment? We have always found a DNase treatment performed on a silca gel memebrane column to be the most effective for eliminating gDNA and void of possible cations that can be left behind and possibly inhibit enzymatic reactions with a post RNA recovery treatment. We recommend to add twice the amount of DNase units (27.3U) and double the incubation period (30 min) with an on-column treatment to insure all or most gDNA has been eliminated. If an “in tube” DNase treatment is used (such as the Turbo Kit by Ambion), be aware that downstream quantitation maybe artificially inflated.
How can I concentrate my RNA? RNA can be concentrated with a speed vac without compromising the integrity if done properly. First, properly clean the speed vac with RNaseZap and Ethanol to insure no RNases are present. On each sample tube install a breather membrane (breathe easier tube membranes) over the tubes to minimize introducing exogenous materials. For instructions on proper speed -vac cleaning, go to the protocols link.
What RNA kit should I use for cleaning or concentrating RNA? This is highly dependent on the starting concentration of the RNA you wish to clean or concentrate. To concentrate or clean total RNA, we prefer silica column technologies (such as RNeasy mini) are acceptable however if you desire miRNA’s or have limited RNA then RNeasy Micro kit is ideal because it can be eluted in as little as 12 ul of 60 C water.
What about extraction of bacteria or yeast? Certainly bacteria and yeast will require additional effort. This includes growing bacteria and yeast to no greater then early log and performing some enzymatic treatment. For yeast we recommend lytic enzyme from Sigma and following the RNeasy protocol. However, it is required to microscopically check the % sphearoplasting under the microscope. Not all lytic enzymes are created equal. For a high efficiency protocol for yeast see: http://vgn.uvm.edu/outreach/Outreach-Lab_Manual-final1-16-06.pdf
It is understood that Gram negative bacteria are easier to get RNA from than Gram Positive. In both cases we recommend a Protinease K and lysozyme treatment followed by 50 C Trizol addition and homogenized using diamond and a the FastPrep-24.
RNA Extraction in the presence of ECM or similar Major problems can occur when isolating RNA using silica columns from unusual matrices such as those with high ECM, fat, or alginate as they interfere and coat the silica and render it inactive. For this purpose the first choice is Trizol followed by a silica clean-up column.
RNA Quantification:
How should I quantify my RNA? The first approach would be to use the NanoDrop Spectrophotometer located in 305 or 307HSRF. 
The dynamic range of confidence for this instrument is 3 ng/ul to 3 ug/ul. It requires 1-2 ul of your sample and generates an absorbance trace that can reveal if other contaminants are present. When working with low recovery targets or samples that have carry over salt or TriZol (which will not generate accurate NanoDrop readings), it is recommended to use the Qubit Spectrofluorometer . Please see a facility staff member for access or training to the facility instrument.
What is a 260/280 ratio? The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm. See Thermo Scientific T042 technical Bulletin for examples.
What is a 260/230 ratio? This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Very pure RNA will indicate an expected 260/230 values in the range of 2.0. However this is concentration dependent and also may depend on the extraction method. Samples that have low concentrations of recovered RNA may have values of 1 and should be fine. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm. EDTA, carbohydrates and phenol all have absorbance near 230 nm. The TRIzol reagent is a phenolic solution which absorbs in the UV both at 230 nm and 270 nm. Guanidine HCL used for DNA isolations will absorb at ~230 nm while guanidine isothiocyanate, used for RNA isolations will absorb at ~260 nm. See Thermo Scientific T042 technical Bulletin for examples.
Other RNA quantification concerns? For samples that are isolated for FACS or LCM or that have other interfering molecules as indicated by the Nanodrop, maybe be quantified using the Agilent Bioanalyzer 2100 and the QuBit flourometer by special arrangement with the core.
RNA Assessment:
Agilent 2100 Bioanalyzer:
RNA quality is essential for gene expression analysis using microarray technology. This system allows for quantitation and quality assessment with small sample volume. As little as 200 pg/ul is required per analysis. RNA degradation is assessed utilizing the 28s and 18s rRNA. RNase degradation is easily detected by a shift in the RNA size distribution towards smaller fragments and a decrease in fluorescence signal of ribosomal peaks.
The microarray facility will run up to 11-12 (11 for a pico and 12 for a nano) samples for $30 using one chip. If you choose to run fewer samples than this, the cost will be still $30.
Why do I need to test my RNA before target preparation submission? This will insure that the sample input is of a quality that will generate usable endpoint data.
What is included in the RNA assessment report? The RNA assessment report includes a trace, RIN number and RNA concentration. The RNA concentration is not reliable, and if needed for LCM, FACS, FNA’s samples, it should be specifically requested on the order form submitted. We will use in-house standards to insure a more accurate RNA concentration reading for those who request this need. The RIN number is an indication of the integrity of the RNA based on using the ribosomal subunits as a surrogate for the intactness of mRNA.
What is RIN? RIN (RNA Integrity Number) is an acronym used to assess the intactness or integrity of your RNA sample. RIN scale range is 1-10, one being the worst and ten the best.
What level of RNA degradation can I have and still move forward with expression analysis? RIN numbers greater than 6 have been considered acceptable in the Real Time PCR community for downstream analyses, but a study by M. Pfaffl (S. Fleige and M. W. Pfaffl. RNA integrity and the effect on the real-time qRT-PCR performance, Mol Aspects Med. 27, 126-139. (2006)) indicates RIN numbers greater than 5 have no appreciable affect on downstream measurements as measured by RT-qPCR. However for microarray, we prefer RINs greater than 7. Samples that are below these values should be reviewed with the staff.
Samples of the RNA quality check with the Agilent 2100 Bioanalyzer
 |
High quality, RIN=9.6 |
 |
Partially degraded, RIN=7.5 |
 |
Degraded, RIN=2.3 |
This is the electropherogram output we obtain from the Agilent 2100 Bioanalyzer. Your samples are analyzed in order to obtain quantitative and qualitative measurements of your RNA extraction. The two distinctive peaks on the left represent the ribosomal subunits 28s and 18s.
Target Preparation (Amplification) for Expression Arrays:
What target preparation is used for expression arrays and why?
3’ arrays: The NuGEN Ovation target preparation system is used if RNA concentration is 5 ng/ul or higher. For lower recovery targets, the NuGEN Pico Ovation is generally employed but special procedures are inline to perform a NuGEN Ovation.
Exon and Gene arrays: The NuGEN WT-Ovation system combined with the WT-Ovation Exon Module has been successful in the facility for generating high quality, reproducible data endpoints. The Facility staff have extensively tested target preparation systems compatible with Affymetrix GeneChip technologies and have found the NuGEN product lines to be most sensitive and requiring very little input material.
Can I still request the standard Affymetrix -Eberwine target preparation? Yes, if requested. It is recommended if you want to generate new data that can be directly compared to a previous data set that used the “Eberwine” approach. The facility will only offer this target preparation for those who specifically request it.
What is a SPIA isothermal amplification? (reprinted directly from NuGEN Technologies , Inc. Bulletin 3)
The Ovation® RNA Amplification Process (3 prime initiated) Step 1. First strand cDNA is produced using a unique DNA/RNA chimeric primer and reverse transcriptase. The DNA portion of the primer contains a poly T sequence which anneals to the 3' poly(A) sequence of each polyadenylated transcript. The RNA portion of the primer contains a unique sequence that is used to incorporate a priming site for the linear amplification step (Step 3 below).
Step 2. Second strand cDNA is synthesized with DNA polymerase. This cDNA strand is in the same sense orientation as the original mRNA. It is important to note that the 3' end of the newly synthesized second cDNA strand will contain the complement to the unique RNA sequence in the first strand chimeric DNA/RNA primer.
Step 3. The isothermal linear amplification step uses a second DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the short RNA portion of the double stranded cDNA revealing a site for binding the second DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3´ end of the primer and displacing the existing forward strand. RNA at the 5´ end of the newly synthesized strand is again cleaved by RNase H partially exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified single stranded antisense (opposite sense) cDNA products.
What is SPIA? Single Primer Isothermal Amplification
What if I only have picogram amounts of RNA? We have amplified as little as 500 pg of total RNA using a modified technique we call double SPIA. The facility staff has been successful in generating enough cDNA for further processing from as low as 60pg/ul RNA input when using 2.5X SPIA reagents.
How do I request a double SPIA amp? This is done by special arrangement.
How long does it take to get my Microarray data after I submit my RNA? If the RNA is submitted according to our sample submission guidelines, we will complete the project in 10 business days or less.
After the Core runs the microarrays, what will I get back for data? Once the microarray data is generated, we will copy the data on the three DVDs and file transfer the data to you. We will keep a disk on hand and two CPU copies. Keep inmind DVD data does not last forever and disks can lose their data in as little as 5 years. SO copy your data to a safe place.
Will data automatically be transferred to bioinformatics dept and analyzed? NO!!! You will need to contact them and arrange to get into there data analysis queue. They will ask you for a defined experimental design in writing. You will be required to interface with them independent of the microarray facility.
