Biomedical Engineering Reference
In-Depth Information
probe concentration at which high saturated specifi c signals is obtained with
low background noise.
4.4. Hybridization
13. A critical consideration of ISHH is stringency, which refers to the condition
favoring dissociation of nucleic acid duplexes; specifi c RNA-RNA duplexes
will withstand high stringency conditions as compared to nonspecifi c hybrids.
Increased stringency can be obtained, for example, by increased hybridization
temperature, increased formamide concentration, and decreased salt concentra-
tion. Most probes hybridize specifi cally to their target mRNAs at a
T
m
(melting
temperature) of - 25°C. Increasing the hybridization temperature can increase
tissue damage; thus it is recommended to mount tissue on Superfrost or poly-
L
-
lysine glass slides. Inclusion of formamide (destabilizes nucleic acid duplexes,
thus lowering the effective
T
m
) allows the hybridization to be carried out at lower
temperatures. Only fresh formamide should be used for the hybridization solu-
tion. Moreover, uneven hybridization signal can be present if the SSC-formamide
solution in the hybridization tray is not fresh.
14. Denhardt's solution and dextran sulfate are important components of the
hybridization buffer. Denhardt's solution, which consists of Ficoll, bovine serum
albumin, and polyvinylpyrrolidone, helps to decrease nonspecifi c binding to
proteins and nucleic acids. The anionic polymer dextran sulfate greatly enhances
the apparent rate of hybridization, thus leading to higher hybridization signals.
Only DEPC-treated distilled water should be used in the hybridization buffer.
15. The addition of DTT is important for
35
S-labeled riboprobes, as it helps to
prevent formation of the disulfi de bond of the
35
S to uracil. Although
33
P does not
have a sulfi de bond, we have found that the inclusion of DTT is still benefi cial for
decreasing background signal. DTT should be weighed under a fume hood and
as such it is best to make aliquots of the DTT and store in a refrigerator, adding
DEPC-treated distilled water only when needed for each experiment.
16. If there are many hybridization trays because of high processing of slides,
then immediately put each tray into the incubator after it is fi lled with the
coverslipped slides before completing the next set of trays. This will avoid
hybridization at low stringency (room temperature) for the sections in trays that
were completed fi rst.
4.5. Post-Hybridization
17. It is important to have the post-hybridization solutions at the proper temperature
before beginning the post-hybridization washes.
18. The RNase A treatment is important because it will digest single-stranded
RNA, essentially removing non- or poorly hybridized labeled probe, leaving the
double-stranded hybridized pairs intact. Sometimes RNase T1 (1 U/mL) can
also be added to the RNase A post-hybridization incubation when the signal-to-
noise ratio for low expressing transcripts is still not optimal. The RNase T1
digests portions of the single-stranded RNA that are not digested by RNase A.
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