DNA and RNA extraction
DNA extraction
High quality DNA is essential for many downstream applications. The type and quality of the material, as well as the intended use of the extracted DNA, decide the choice of method.
For formalin-fixed and paraffin-embedded tissue sections, two methods are currently in use for extracting DNA, either a modified version of the QiaAmp DNA mini-kit, or a manual phenol/chloroform procedure.
For fresh, or fresh frozen, blood, tissue samples, and cell lines, large amounts of DNA is extracted by a semi-automatic phenol/chloroform procedure (Applied Biosystems, ABI 341 nucleic acid purification system). This type of extracted DNA can be stored for 10-15 years at 4°C with maintained optimal quality.
DNA may also be extracted with the Qiagen AllPrep kit (see 'b)RNA extraction with Qiagen AllPrep kit' below) using Qiagen AllPrep DNA spin columns, or from the Trizol phase after RNA extraction (see 'a)RNA extraction with Trizol' below) using a modified phenol/chloroform-method.
For all methods, the quality and concentration of the purified DNA are measured using a NanoDrop spectrophotometer (fig1) and a Fluorometer.
High quality DNA is essential for many downstream applications. The type and quality of the material, as well as the intended use of the extracted DNA, decide the choice of method.
For formalin-fixed and paraffin-embedded tissue sections, two methods are currently in use for extracting DNA, either a modified version of the QiaAmp DNA mini-kit, or a manual phenol/chloroform procedure.
For fresh, or fresh frozen, blood, tissue samples, and cell lines, large amounts of DNA is extracted by a semi-automatic phenol/chloroform procedure (Applied Biosystems, ABI 341 nucleic acid purification system). This type of extracted DNA can be stored for 10-15 years at 4°C with maintained optimal quality.
DNA may also be extracted with the Qiagen AllPrep kit (see 'b)RNA extraction with Qiagen AllPrep kit' below) using Qiagen AllPrep DNA spin columns, or from the Trizol phase after RNA extraction (see 'a)RNA extraction with Trizol' below) using a modified phenol/chloroform-method.
For all methods, the quality and concentration of the purified DNA are measured using a NanoDrop spectrophotometer (fig1) and a Fluorometer.
RNA extraction
RNA from cell lines, fresh frozen human tissue, or human tissue processed in "RNAlater" is extracted using two different methods. The available amount of material guides the choice of method.
a)RNA extraction with Trizol
RNA is extracted with Trizol reagent using the standard RNA extraction protocol from Invitrogen.
b)RNA extraction using Qiagen AllPrep kit
RNA is extracted using the standard protocol included in the Qiagen kit.
The two methods are both easy to perform. The concentration and purity of the RNA is measured using a NanoDrop spectrophotometer(fig 2), and the quality of the RNA is evaluated by BioAnalyzer measurements (fig 3).
RNA from cell lines, fresh frozen human tissue, or human tissue processed in "RNAlater" is extracted using two different methods. The available amount of material guides the choice of method.
a)RNA extraction with Trizol
RNA is extracted with Trizol reagent using the standard RNA extraction protocol from Invitrogen.
b)RNA extraction using Qiagen AllPrep kit
RNA is extracted using the standard protocol included in the Qiagen kit.
The two methods are both easy to perform. The concentration and purity of the RNA is measured using a NanoDrop spectrophotometer(fig 2), and the quality of the RNA is evaluated by BioAnalyzer measurements (fig 3).