It is not impossible to extract RNA from it, but it involves quite a lot difficulty and the RNA yield is very low that it is only suitable for RT-PCR. The preserved conditions of the paraffin-embedded tissue are most critical. First, if the tissue had been fixed for more than 24 hours, RNA experienced modification that cannot be used for RT-PCR anymore. Second, the older the tissue block is, the poorer the RNA yield is. Tissue blocks stored for more than 6 months are thus not suitable. Third, only short-template RT-PCR (< 400-bp) can be performed. Fourth, since only a tiny bit of tissue sample is processed, it is of course hard to disrupt the sample mechanically. In this case, Proteinase K should be used to digest the sample. However, Proteinase K is not provided in the kit. Fifth, since total RNA extracted is so little, RNA carrier (like the one used in Viral RNA Extraction System) should be added to enhance RNA recovery. Sixth, paraffin needs to be removed before the tissue is to be disrupted. Viogene Total RNA Extraction System is not tuned for processing paraffin-embedded tissues.

No, some genomic DNA (and plasmid DNA, if present) can be co-purified with RNA. DNA can be removed by adding RNase-free DNase I to the RNA sample. DNase I can then be removed by phenol/chloroform extraction.

1) Poor yield of total RNA is mostly due to incomplete sample lysis, thus leading to incomplete release of RNA. Since good yield and good quality of total RNA are only assured when sample is properly handled and lysed completely, do not use more than the amount of sample suggested in the protocol.

2) Thorough celluar disruption is critical for high RNA quality and yield. RNA that is trapped in intact cells is often removed with cellular debris and is unavailable for subsequent isolation. Therefore it is crucial to choose the disruption method best suited to a specific tissue or organism to maximize yield. Mechnical cell disruption techniques include grinding, homogenization with douce or rotor-stator homogenizers (polytron), vortexing, sonciation, and use of bead and freezer. Complete disruption of some tissues may require using a combination of these techniques. Rotor-stator homogenizers, alone or in conjunction with other disruption techniques, generally result in higher RNA yields than other types of homogenizer.

3) Another most frequent cause of low RNA yield is overloading the column, which can cause the column to clog or can prevent the RNA from binding to the membrane efficiently. Methods that reduce viscosity, such as reducing sample amount, diluting the viscous lysate with RX Buffer, disrupting the sample more extensively, and centrifuging to remove insoluble remains, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting loading into two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery.

4) When RNA is to be eluted, make sure that RNase-free ddH2O is added onto the membrane and penetrate into it. If ddH2O still retains on the membrane, pulse centrifuge the column for a few seconds to drag it into the membrane.

5) Eluting the column twice can result in a higher RNA recovery, especially when expected RNA yield is more than 30 mg.

Three critical steps, if not done well can cause RNA degradation. They are 1) handling and storing of samples, 2) disruption of samples, 3) storage of eluted RNA.

1) Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissecting. If samples cannot be processed immediately, it should be flash frozen in liquid nitrogen and stored at -80°C. Samples should be handled with RNase-free tools.

2) When sample is disrupted, disruption needs to be fast and thorough. Slow disruption, e.g. placing cells or tissue in RX Buffer without any additional physical shearing, may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in RX Buffer.

3) After elution of RNA with RNase-free ddH2O provided in the system, store RNA at -80°C.

Degradation of RNA may also occur during loading into a gel, use gel and fresh running buffer prepared using DEPC-treated ddH2O as well as properly cleaned gel tray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining.

Yes, PX Buffer does not affect the chemically linked DIG on dNTP. Similarly, this system can be used to clean up ³²P-labeled DNA fragment.

Both systems can be used to clean up DNA fragments (100-bp to 10-kb) from enzymes, salts, and dNTPs. PCR Advanced^TM Clean Up System is a cheaper choice for cleaning up DNA fragments in solution. When a specific DNA fragment is to be purified, gel electrophoresis is needed. A gel slice containing the desired DNA fragment is excised, and DNA is extracted using Gel Advanced^TM Gel Extraction System.

It is not recommended to use this system to clean up sequencing reaction because PCR products smaller than 100-bp cannot be recovered effectively, thus making reading of the first 80-100 nucleotide sequence unfeasible.

When the pH of the enzymatic reaction solution is higher than 7.5, DNA recovery will be reduced. In this case, add 10 µl of 3M potassium acetate of pH 5.0 to the DNA solution before adding PX Buffer.

PCR-M^® Clean Up System can only effectively (>90%) removes primers of less than 40-bp. When primers or dimer products are of more than 40-bp, they cannot be effectively removed. In this case, separate the PCR product from the dimer products by electrophoresis, excise the gel slice containing the desired product and purify it using Viogene Gel-M^® Gel Extraction System.

There are several possible reasons:

1)      Do not overload the column with too much DNA. Higher recovery is attained when lower amount of DNA is loaded. Split loading high amount of DNA into more than one column.

2)      If ddH₂O is used for elution, make sure than its pH is between 7.0 and 8.5. pH lower than 7 leads to lower elution efficiency.

3)      Make sure that complete DNA elution takes place by adding no less than 30 ml of elution solution onto the membrane and letting it completely absorbed into the membrane before centrifugation.

4)      Large DNA fragment is eluted less readily than small DNA fragment. When the DNA product is larger than 5-kb, use elution solution preheated to 60˚C.

The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to that elongation of the PCR product is not complete or that PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA by incubating the solution at 95˚C for 2 minutes and let it cool slowly to room temperature. The re-annealed PCR product can be used as usual in all downstream applications.

<For Gel Extraction>
Add 0.25 volume of isopropanol of the mixture from STEP 3. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp.

<For PCR Clean up>
Add 0.25 volume of isopropanol of the mixture from STEP 1. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp.

The difference between Blood & Tissue Genomic DNA Mini and Blood Genomic DNA Midi and Maxi is that the Midi and Maxi kits do not have LYS Buffer. LYS Buffer in Blood & Tissue Genomic DNA Mini is mainly used for sample digestion of tissue samples. This means that any sample, which only needs EX Buffer for sample digestion as listed in Blood & Tissue Genomic DNA Mini, can be used in the Midi and Maxi kits. These samples include whole, buffy coat, serum, plasma, body fluid, lymphocytes, animal cells, bacteria, viral DNA from blood or body fluid, and integrated viral DNA in animal cells. Follow the Blood & Tissue Genomic DNA Mini protocol for the procedure but use the time duration and buffer volumes as suggested in that of Midi and Maxi kit.

If it is blood stain (dried blood) on a piece of filter paper, it is still OK to use our Blood & Tissue Genomic DNA Extraction Miniprep System to extract DNA from it. What Dr. K. Nobuto can do is to cut the filter paper into small pieces (about 10 mm2). Place 1 to 4 pieces (depends on how concentrated the blood stain is) into a clean 1.5-ml eppendorf tube. Add 20 ml Proteinase K and 200 ml LYS Buffer into the sample. Mix immediately by vortexing for 20 seconds. Follow the Tissue Protocol from step 3.

Several points should be noted to avoid DNA degradation:

1) DNA degradation occurs when the sample is not fresh or is stored improperly for a long time. Samples not used immediately should be flash frozen in liquid nitrogen and stored at -80°C. Genomic DNA in samples stored at room temperature, 4°C, or -20°C are subjected to degradation. It is also not advised to keep samples in buffer or medium and stored at -80°C.

2) For whole blood samples, if they are stored at room temperature for more than 2 days or at 4°C or -20°C, genomic DNA isolated appears smearing at an extent proportional to the storage time.

3) Use fresh TAE or TBE running buffer for electrophoresis, repeatedly used running buffer may be contaminated with DNase.

4) If isolated DNA needs to be stored for a long time, use 10 mM Tris-HCl (pH 9.0) or TE for elution. ddH2O is not advised in this cause because DNA fragments in H2O suffer from gradual degradation through acid hydrolysis readily.

5) If DNA is to be used frequently, elute in 10 mM Tris-HCl (pH 9.0) or TE and store at 4°C. Keep DNA at -20°C only for long-term storage. Repeated freeze-thaw cycles can cause shearing of genomic DNA.

6) Genomic DNA extracted from paraffin-embedded tissue is usually degraded. It is because genomic DNA in paraffin-embedded tissue unavoidably suffers from degradation when sample was treated and stored for a long time. DNA in this case is not suitable for Southern blotting and restriction analysis due to the smearing. However, it is applicable for PCR.

This indicates that RBC or hemoglobins have not been completely lysed or digested. During incubation for Proteinase K digestion, the sample should be mixed every 3-5 minutes by vortexing and inverting to completely disperse Proteinase K and samples.

The key is to use fresh sample and not to overload the column. Low yield or purity of genomic DNA is usually due to incomplete digestion or incomplete lysis of the sample. Starting with a maximum amount or volume of samples does NOT usually give the best yield of DNA. On the contrary, it usually results in incomplete sample lysis and degradation of proteins, thus making extraction of all DNA from the sample unfeasible. Further, it always requires subsequent removal of undigested residues and yields viscous sample lysate. When the lysate is too viscous, it not only has difficulty in passing the column, but also indicates the presence of an abundant amount of contaminants such as proteins and salts. Contaminants of high amount not only affect DNA binding, but also may not be washed off completely, leading to carry over to the eluted genomic DNA. Therefore, a good quality and yield of DNA is only expected when a sample is completely digested. We advise starting with half of the maximum amount of sample suggested. When there is no problem in digesting the sample completely and passing the lysate through the column, amount of the sample to be applied can be increased gradually in the subsequent preparations.

If a sample is rich in protein, e.g. fish flesh, complete digestion will not be achieved using the amount of Proteinase K and buffer suggested in the protocol. If a sample cannot be digested completely or appears very viscous, add more LYS Buffer and repeat incubation. Centrifuge the sample at full speed for 5 minutes to remove undigested remains and only use the supernatant in the following steps. In the subsequent preparations, a lower amount of the sample should be used. A general rule of thumb is to start with half of the maximum amount of sample suggested. When there is no problem in digesting the sample completely and passing the lysate through the column, amount of the sample to be applied can be increased gradually in the next preparations.

Genomic DNA extracted from paraffin-embedded tissue is usually degraded. It does not show as a clear strong band of about 20-30 kb as we usually see when fresh sample is used. It is because genomic DNA in paraffin-embedded tissue unavoidably suffers from degradation when sample has been treated and stored over a long period. DNA in this case is not suitable for Southern blotting and restriction analysis due to the smearing. However, it is applicable for PCR.

For yeast, use Blood & Tissue Genomic DNA Extraction System. For filamentous fungi and mushroom, use Plant Genomic DNA Extraction System