SileksMagNA™ FFPE DNA/RNA Isolation kit
The kit contains a sufficient amount of reagents (except of deparaffinization agent - xylene or toluene) for:
100 procedures for isolation of either DNA or RNA
50 procedures for combined isolation of DNA and RNA
- Application
- Characteristics
- Composition
- Isolation features
- Downloads
- Additional
The kit is designed for efficient and fast isolation of DNA and / or RNA from paraffinized samples fixed in formalin (FFPE - formalin fixed paraffin embedded).
The kits include all neccessary reagents and buffers except a deparaffinization agent (xylene or toluene should be used).
The number of reagents in the kit is sufficient for 100 procedures for isolating either DNA or RNA.
It is also possible to simultaneously extract DNA and RNA from one sample at the same time. These will be two isolation procedures. In this case, the total number of isolations by the kit is reduced to 50 procedures.
- Number of procedures100 procedures for isolation of either DNA or RNA
50 procedures for combined isolation of DNA and RNA - Starting materialformalin fixed paraffin sample (FFPE) section
- Starting amount of material1-5 slices 10 mkm thick
- Approximate yielddepending on the source material
- Storage+4 °C
proteinase is recommended to be stored at -20 ° C - Transportationno special conditions required, but Proteinase is recommended to be transported at +4°C
Proteinase, 1 ml
Proteinase buffer, 1-fold, 10 ml
START buffer, 12 ml
Lysis & Binding buffer, 24 ml
SileksMagNA magnetic particles, 1 ml
Wash 1 buffer, 30 ml
Wash 2 buffer, 30 ml
Wash 3 buffer, 30 ml
Final Wash buffer, 30 ml
Elution buffer, 10 ml
RNA Protector, 0.5 ml
The features of the шыщдфtion from paraffinized samples fixed in formalin and subsequent work with the isolated material is that in the process of fixation and paraffinization, the nucleic acid (DNA and RNA) in the samples undergoes strong fragmentation and chemical modification with formaldehyde. As a result, nucleic acid (NA) isolated from paraffinized samples is most often a highly fragmented NA, which is fundamentally different from NA isolated from fresh or frozen samples. The effect of formaldehyde treatment affects not only the complexity of NA isolation, but also subsequent procedures involving various enzymes. Formaldehyde modifications and traces of formaldehyde itself have a strong inhibitory effect on many regularly used enzymes, including thermostable polymerases used in polymerase chain reaction (PCR).
It is often impossible to determine the quality of RNA isolated from paraffinized samples in the gel by the presence of 18S and 28S ribosomal RNA bands. The 18S and 28S rRNA bands are weak or highly smeared, and may be completely absent at worst.
Depending on the quality of the samples, the size of NA can vary greatly. The average size of RNA recovered from paraffinized samples is about 100 nucleotides. The size of DNA fragments can be several times larger, sometimes up to 500 nucleotides.
The quality of the samples and the integrity of DNA are determined by various factors:
- type of tissue,
- storage time of the sample,
- fixing conditions and preparation conditions (pH of the buffer used, temperature, quality of reagents),
- storage conditions of the sample.
The NA isolation procedure proposed in this kit significantly reduces the amount of cross-linking generated during formaldehyde fixation. Depending on the quality of fixation procedure and the age of the sample, the length of isolated nucleic acids may vary in a very wide range. It is very important to take it into account when designing reliable PCR system.
VERY IMPORTANT !!!
We recommend to avoid designing primer systems that amplify fragments larger than 100 bp. For reverse transcription it is recommended to use specific or random primers and avoid using oligo(dT) primers.