Frequently Asked Questions about RNA, gDNA, & cDNA
Below you will find some of the questions that we are frequently asked concerning use of RNA and gDNA. If you have a question that is not listed below, please contact us.
Should I use RNA or gDNA as a template for amplification?
About half of the papers published so far for immune repertoire sequencing use genomic DNA samples, while the other half use RNA. Although we have primers that work for both sample types, we prefer RNA as a starting template, if possible.
The main concern about using genomic DNA is the inclusiveness. There is only a certain amount of template DNA that one can add to a PCR reaction. Each diploid human cell may have about 6.6 pg of genomic DNA of which the rearranged V(D)J represents a very small portion. So, if 100 ng of genomic DNA is used in the reaction, this represents only about 16,000 cells, resulting in a restricted view of the repertoire. Although more genomic DNA can be used, there is a limit to how much can be added. In contrast, the cDNA template created from iRepertoire’s RNA reagent systems will be immune specific, maximizing the amount of desired template included in the reaction, and excluding unnecessary and potentially disruptive gDNA.
A second concern about using genomic DNA as a template to generate a sequencing library is the background. After the V(D)J rearrangement, those V and J segments not involved in the recombination will still be in the genome and can serve as perfect binding sites for the primers. Binding at these sites will generate background amplifications, exhaust primers, and introduce bias.
Those who prefer to use genomic DNA have a variety of valid reasons. Samples are much easier to obtain, and even biopsied samples from tissue or slides can be used. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. However, one can also argue that the RNA expression level of the T and B cell receptors may reflect the functional status better.
So, should you use gDNA or RNA? The answer really depends on your research goals. If the repertoire changes you are looking for will be represented by dominant clones, then both gDNA and RNA will work. If, however, you need to see the broader diversity, RNA may be better.
Can I use cDNA as a template?
We recommend using our primers to create cDNA rather than using poly dT primers or random hexamer primers for several reasons. By creating the cDNA using iRepertoire’s Reagent Systems, the cDNA library will have an increased concentration of immune specific cDNA relative to a library produced from catch-all primers. These non-specific cDNA libraries will include cDNA sequences from any expressed protein in the cell, reducing the amount of immune specific cDNA template available for amplification. Furthermore, the creation of a robust cDNA library using non-specific primers requires a lot of input RNA. We can reduce the amount of input RNA down to a recommended 100 ng from sorted cells(however, less may also work) because the cDNA library produced will be immune specific.
My cells are not sorted; therefore, I have total RNA from whole blood or PBMCs. Should I use more RNA in my reaction?
Yes, we would recommend using more RNA if possible. Please keep in mind that 100 ng is the minimum recommended starting material, but you will likely increase the diversity of the library if you use more input RNA (not exceeding the maximum input of the Qiagen One-Step RT-PCR kit (≤ 1µg per 25 µL reaction)). When RNA from sorted cells is used, we typically find that we can use less starting template. When using total RNA from whole blood or PBMCs, we recommend using the maximum input of the Qiagen One-Step RT-PCR kit.
I have RNA with RIN 8.1 and its concentration is 6 ng/µL. For the PCR1, it recommends minimum 100 ng of template. The protocol suggests 25 µL reaction volume which gives 13.75 µL as max volume of template to be added. This makes my template 82.5 ng for the reaction which is less that 100 ng. Would it be wise to run this sample?
In our experience, if the RNA concentration measurement that you have provided is accurate, we are typically able to get amplification. Therefore, we suggest going ahead and trying to amplify the sample. We have had good success with similar amounts of RNA. If, however, this measurement includes carrier RNA, then it is less likely you will get amplification because the actual sample concentration is likely to be lower. Alternatively, you could concentrate the sample; however, this will result in some loss of sample, and there is increased risk of RNase contamination. Therefore, we recommend first trying the amplification.
How can I stabilize RNA from cells or tissue?
If RNA extraction cannot be performed immediately, isolated cells should be stored in Qiagen® RNAprotect cell reagent (www.qiagen.com catalog #76526) at 2-8°C for up to 1 week. Tissue samples should be stored in Qiagen® RNAlater (www.qiagen.com catalog #76106) at 2-8°C for up to 4 weeks.
What type of kits do you recommend for RNA extraction?
Human and Mouse cells or tissue: Qiagen® RNeasy Mini kit (www.qiagen.com catalog #74104)
Human and Mouse cell samples with low counts: Qiagen® RNeasy Micro kit (www.qiagen.com catalog #74004)
Human and Mouse tissue: Norgen Animal Tissue RNA Purification kit (www.norgenbiotek.com catalog#25700)
How should I store RNA?
Purified RNA may be stored in RNase-free water at -20°C for one month or -80°C for up to one year. Under these conditions, no degradation of RNA should be detectable. To avoid multiple freeze-thaws of RNA, we recommend that you aliquot it into smaller lots prior to freezing.
How do I quantify RNA/DNA?
The concentration of RNA/DNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Small amounts of RNA/DNA may be quantified using an Agilent® 2100 Bioanalyzer or by fluorometric quantification.
How do I use carrier RNA?
Carrier RNA reduces the loss of sample RNA during the extraction process. Carrier RNA should be added prior to homogenization of cells when fewer than 500 cells are present. The Qiagen® RNeasy micro kit, which contains carrier RNA, is recommended for RNA extraction from small numbers of cells.
Which sequencing platform should I choose?
The choice between Illumina and Roche 454 is project dependent. The Illumina HiSeq platform provides many reads, and therefore, more samples can be pooled per lane than Roche 454. Overall, Illumina sequencing is cheaper than Roche 454, however, the read length is shorter, approximately 150 bp. Therefore, the Illumina platform is recommended for TCR sequencing and provides CDR3 data.
Roche 454 sequencing is recommended for B cell studies because the read length is longer, approximately 350 bp with sequence information beginning from within the first framework region and extending into the beginning of the C-region. The trade-off for increased sequencing length with Roche 454 is reduced sequencing depth (ie. fewer available reads) when compared to using the Illumina system. In addition, Roche 454 sequencing is more expensive than Illumina, and fewer samples should be pooled per run (due to the reduced availability of reads). However, if you are expecting your library to lack diversity (ie. clonal expansion of just a few clones), then pooling more samples is possible. The longer read length of the 454 platform allows for the determination of the class of the antibody. Furthermore, for those studies where hypermutation information is essential, the Roche 454 technology is the only choice currently.
What is the expected RNA yield from lymphocytes?
1 x 10^6 unstimulated lymphocytes yield approximately 0.5 μg total RNA. RNA yields may vary depending on the cell type and the activation state of the cell.
How should I handle the RNA and/or DNA?
Follow the recommendations below to obtain the best results:
• Always work quickly when dealing with RNA, as it is extremely sensitive.
• Maintain a sterile environment when handling nucleic acids (NA) to avoid any contamination from nucleases.
• Ensure that no nucleases are introduced into the solutions supplied with the reagent system.
• Make sure that all equipment coming in contact with NA is sterile, including pipette tips and tubes.
• RNA is extremely sensitive to degradation. Ensure that RNA is stored at -80˚C prior to use and maintained at 4˚C during template addition. Gently flick RNA sample to mix once thawed, DO NOT VORTEX.
• RNA or DNA should be thawed while on ice.