FAQ

Q1. What does the DEG stand for?

A1. The DEG stands for Differentially Expressed Gene. The DEG can be found in two or more RNA samples from any organism to be compared. The DEG study has been widely activated in the various biological fields, to be specific, for studying DEGs related to specific disease such as tumor, drug treatment, different developmental stages, environmental change like stress and etc.

Q2. What is the function of regulator in the structure of ACP?

A2. The arbitrary ACP comprises: (a) a 3'-end arbitrary nucleotide sequence, (b) a 5'-end non-target universal nucleotide sequence, and (c) a regulator bridging the 3'- and 5'-end portions. In particular, during the first annealing temperature, the regulator positioned between the 3'- and 5'-end portions in the structure of ACP switch off the function of the 5'-end portion at such hybridisation temperature but switch on only the function of the 3'-end portion. Eventually the regulator excludes the 5'-end portion from annealing to the template at the first annealing temperature whereas the 3'-end arbitrary sequence exclusively anneals to the template, leading to dramatically increasing the strength of the specific annealing of the 3'-end core portion. During the second annealing temperature, the arbitrarily primed products only will be amplified by using the 5'-end portion of the ACP as a priming site server. Therefore, the function of the regulator is to control an annealing portion of ACP in association with annealing temperature so that it plays a key role in maximising primer annealing specificity.

Q3. Is the one RT reaction of the GeneFishing™ DEG Premix Kits able to obtain 100% of cDNAs?

A3. Yes, the use of the dT-ACP used in RT results in only one cDNA population by one reaction, which covers 100% of cDNA population. The conventional DD-PCR generates many PCR products including a high percentage of false products. Thus, in order to reduce the number of amplified PCR products, the conventional DD-PCR requires at least 3 to 12 different RT reactions with each different anchor dT primer and each RT reaction generates a sub-population of cDNA pools. In contrast, GeneFishing™ PCR produces a few PCR products and thus does not require sub-populations. Therefore, GeneFishing™ DEG Premix Kits greatly increase the efficiency of the method by generating a substantially standard pool of single-stranded cDNA from each experimental mRNA population.

Q4. How is the resolution of agarose gels for the detection of DEGs having small changes in expression levels?

A4. Annealing Control Primer (ACP) incorporated in GeneFishing™ DEG Premix Kits dramatically improves specificity of PCR amplification and results in a few PCR products. In addition, GeneFishing™ Technology allows the use of the sufficient amount of starting material and the high concentration of dNTP. Eventually, these benefits allow the ethidium bromide-stained agarose gel to detect DEGs. All PCR products obtained from GeneFishing™ PCR can be detectable by Northern blot analysis. Thus, the bands shown on agarose gels by GeneFishing™ Technology have sufficient resolution to be detected by Northern confirmation. Even small changes in expression levels which are detected on PAGE gel are also detected on agarose gels without any resolution difficulties.

Q5. What range of PCR products is generated and cloned by the GeneFishing™ DEG Premix Kit?

A5. Each GeneFishing™ PCR reaction generates long-distance PCR products ranging from 100 bp to 2 kb which can be detected on agarose gels. This wide range of coverage increases the chances to find DEGs and also provides more significant sequence information for the prediction of gene function. In contrast, the current differential display methods generate short cDNA fragments (typically 100-500 bp) which correspond to the 3'-end regions of the genes and thus usually do not contain a large portion of the coding region. Therefore, the labour-intensive full-length cDNA screening is needed unless significant sequence homology, informative for gene classification and prediction of function, is obtained. In addition, the sequencing PAGE gels cannot discriminate over 500 bp fragments.

Q6. Can the arbitrary ACPs bind to any genes in any organisms?

A6. Yes, each of 20 different arbitrary ACPs comprises a randomly selected 10-mer core sequence. Thus, if cDNAs in humans have complimentary sequences against the arbitrary ACPs, each arbitrary ACP binds to the cDNA templates at the first-stage PCR and then arbitrarily primed products are amplified at the second-stage PCR. During the first-stage PCR, the core sequence (10-mer) of the arbitrary ACP hybridises to the first-stranded cDNAs with at least 8-mer (1 of 22 clones) base pair matches, usually 9-mer (10 of 22 clones) to 10-mer (11of 22 clones) base pair matches. For this reason, fewer bands per reaction are detectable as compared to current differential display methods, but all of the bands differentially expressed on agarose gel obtained from the GeneFishing™ PCR are real and authentic DEGs.

Q7. How many DEGs are detectable on agarose gel using the GeneFishing™ DEG Premix Kits?

A7. The number of clearly visible bands resolved on each agarose gel by the GeneFishing™ DEG Premix Kits varies between 5 to 15 depending on the types of samples ranging in size from 100 bp to 2.0 kb. However, each amplified product corresponds to a single mRNA and each DEG is real, authentic. Although current differential display methods generate about 50 to 100 bands, the conventional arbitrary primers often mismatch and hybridise to distinct regions of the same cDNA, so that the amplified products are occupied by significant percentage of false positives and also different positive clones might correspond to the same gene. Thus, the current differential display methods lead to intensive workload in identifying true positive DEG candidates. In contrast, the use of ACP system enables the researchers to avoid this redundancy so that such subsequent labour work can be minimised. Although, fewer bands per reaction are detectable as compared to the current differential display methods. This drawback can be overcome by using more arbitrary ACPs.

Q8. How many reactions can be performed using GeneFishing™ DEG Premix Kits regarding to samples? How many samples can be compared at the same time?

A8. Depending on the type of GeneFishing™ DEG Premix Kits, the numbers of reactions are different. For example, GeneFishing™ DEG Premix Kits 101 ~ 106 each mainly comprise 20 different arbitrary ACPs (Annealing Control Primers) and dT ACP and each arbitrary ACP is provided for 20 reactions. Thus, if 2 samples are to be compared to identify DEGs between them, ten times of GeneFishing™ PCR reactions can be repeated with the two samples. If 4 samples are to be compared, five times of GeneFishing™ PCR reactions can be repeated with the 4 samples.

Q9. We have a little amount of RNA. Is it possible to use 2µg of total RNA as starting material in the GeneFishing™ DEG method?

A9. Yes, when we performed reverse transcription using mouse conceptus total RNA to confirm limitation of total RNA, reproducible results could be obtained by using at least 250ng of total RNA limitation. In this experiment, the step of DNAse I treatment was omitted.

Q10. Can I use this kit with plant RNA?

A10. Yes. GeneFishing™ DEG Premix Kits can be used for any eukaryotic organisms including human, animal, plant, fish and so on. We recommend to use 3µg of total RNA as starting material from each source.

Q11. What is the minimum difference to be detected on agarose gel in expression of a particular target gene between the test and reference samples?

A11. At least 1.5-fold difference in expression levels can be detected on agarose gel and also confirmed by Northern blot analysis.

Q12. Is there any tip in using a "hot start" PCR in your GeneFishing™ PCR?

A12. For the best results, hot start PCR technique is highly recommended in which the procedure is to set up the complete reactions without the DNA polymerase and incubate the tubes in the thermal cycler to complete the initial denaturation step at >90?. Then, while holding the tubes at a temperature above 90?, the appropriate amount of DNA polymerase can be pipetted into the reaction. Alternatively, you can prepare the reaction mixture (including Taq DNA polymerase) on ice and then thoroughly mix the mixture on ice. Set the sample tubes in a preheated (>90?) thermal cycler and start thermal cycling immediately.

Q13. How do I verify DEGs after cloning?

A13. The DEGs can be verified by Northern blot analysis or RT-PCR.

Q14. Do you have any preference in procedure for cloning of the differentially expressed bands?

A14. We follow general procedure in the general lab. When you extract DEG bands from the gel, we recommend using a method using beads not columns. We use Geneclean II kit from Q.BIOgen (Cat. No. 1001-400) for gel extraction and TOPO TA cloning kit from Invitrogen (Cat.No. K4500-01) for TA cloning. Also when the intensity of interesting bands is not sufficient for direct TA cloning, you can re-amplify the PCR products using our ACP primers or universal primers (Refer to Appendix B in DEG Kits user manual).

Q15. What is the difference between DEG101 and any other DEG Kit(DEG102~106)?

A15. All of the GeneFishing™ DEG Premix Kits (DEG101~106) comprise a different combination of 20 randomly selected arbitrary ACPs, so that each DEG Kit works equally for your target samples. For the first trial of the DEG Kit series, we recommend the DEG101. For further DEG study, we recommend any of the other DEG series(102~106).

Q16. What can I do if the band intensity of DEGs in my sample is very weak?

A16. First, you may have a problem with RT reaction or RNA quality. Make sure that Seegene's dT-ACP1 is added in the RT reaction and check the integrity of the RNA samples by formaldehyde agarose gel. Otherwise, some Taq DNA polymerase may not be compatible with GeneFishing™ PCR. In this case, try again the GeneFishing™ PCR using the recommended Taq DNA polymerase.