Sample quality is the single most important factor in the success of any genomics experiment. High quality input sample yeilds the highest the quality of output data. Any isolation protocol/product may be used that results in pure, intact nucleic acid free of excess salts,proteins, and other contaminants.
The core has a Gentra Autopure LS instrument and is capable of isolating genomic DNA from up to 10mL of blood.
We are able to start with 2ml to 10mL of fresh whole blood, or frozen (comprimised) whole blood.
Current Recharge Rates:
|Whole Blood - 2ml - 5ml:
Compromised Whole Blood - 2ml - 5ml:
Whole Blood from Lysate:
Compromised Whole Blood from Lysate:
| $16.50 /sample
For end user prepared DNA, here are the core's suggestions:
• Is double-stranded.
• Has undergone a minimum of freeze-thaw cycles.
• Has not been exposed to high temperatures.
• Has not been exposed to pH extremes (< 6 or > 9).
• Has an OD 260/280 ratio of approximately 1.8 to 2.0.
• Does not contain insoluble material.
• Does not contain RNA.
• Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.
• Does not contain chelating agents (e.g., EDTA)
• Does not contain divalent metal cations (e.g., Mg2+)
• Does not contain denaturants (e.g., guanidinium salts, phenol), or detergents (e.g., SDS, Triton-X100).
• Does not contain carryover contamination from the starting organism/tissue
When isolating RNA using an organics based method (like Trizol), a two-step purification protocol is recommended since a single organic phase extraction does not adequately remove all contaminants. The second phase of purification is usually best done through a column purification with a DNAse treatment step included.
A commonly used, inexpensive RNA purification protocol is a basic Trizol extraction followed by a Qiagen RNeasy column . There are also many kits available to isolate high-quality RNA.
If using a purification column only, please follow the respective kits protocol. The core also suggests NOT using pipett mixing as a method of homogenization. Please use a small gauge needle instead.
Characteristics of RNA that will perform well:
•Should be intact and non-degraded
•Have an OD 260/280 between 1.8 - 2.0 (a value of 2.0 is generally accepted as pure RNA)
•Have an OD 260/230 between 1.8 - 2.2 (this ratio is an alternative measurement of nucleic acid purity)
•Have a 28S to 18S ratio of 1.0 or greater
•Have a RIN (Agilent's RNA Integrity Number) of 7.0 or higher
•Be pure and free of salts, proteins, DNA, or RNAses that will interfere with reactions or degrade RNA
•Meet the minimum concentration requirements
•Diluted in water, 10mM Tris, ph 7.5 or Qiagen EB buffer
A review paper - RNA integrity and the effect on the real-time qRT-PCR performance
For the services assessing microRNA's (and other small RNA's), it is ideal to isolate total RNA containing the microRNA fraction.
The core does not require the use of any particular method for isolating total RNA containing microRNA and the general guidelines listed above should be observed. We have had customers sucessfully use Qiagen's miRNeasy kit and Invitrogen's mirVana kit for their microRNA isolations.