Biomedical Engineering Reference
In-Depth Information
To make 10× PBS for 1 L, add 800 mL of dH
2
O to a large beaker.
While the water is stirring with a magnet on stir plate, add the following:
80 g NaCl
2 g KCl
29 g Na
2
HPO
4
-12H
2
O
2 g KH
2
PO
4
Finally, fi ll up to 1 L.
*3: Preparation of 2× SSPE:
Ten times dilution from 20× SSPE. For making 5 L of 20× SSPE solution,
mix 876.5 g of NaCl, 153.5 g of NaH
2
PO
4
-2H
2
O, and 37 g of EDTA, add
32.5 mL of 10 N NaOH, and fi ll up to5 L of dH
2
O (no need to use
DEPC-H
2
O).
Store at RT.
*4: The incubation time in 4 % paraformaldehyde/1× PBS is critical for fi xation,
a condition that affects hybridization effi ciency later.
*5: Preparation of 1 L of acetylation solution:
Mix 13.6 mL triethanolamine in1L dH
2
O (no need to use DEPC-H
2
O).
Right before you're ready to use it, add 2.52 mL of acetic anhydride, mix
well, and immediately pour the solution over the slides.
*6: These alcohol solutions can be reused at least 5-10 times, when processing
~60 slides each time.
*7: The amount of hybridization solution depends on how many tissue sections
are attached on the glass slide.
*8: Mineral oil [Sigma, cat#330760] is used.
*9: Hybridization temperature: We recommend 65 °C as a default of hybridiza-
tion and wash temperature. If high background signal was observed as a
result of an RNA probe size (over 3 kbp) or high G/C % (over 70 %), it would
be good to try 70 °C. Conversely, if a researcher wants to try cross-species
hybridization with RNA probes generated from a different species' cDNA
sequence and does not obtain a strong enough, high-quality signal, then try
lower hybridization and wash temperatures in steps of 5 °C (However, in
such case, cloning and using the species-specifi c cDNA fragment of GOI is
the better approach.).
*10: Hybridization time is one of the most crucial points to affect signal intensity
and noise ratio.
We recommend using a standard hybridization time (−15 h) across experiments,
if a researcher wants to compare the results from experiments performed on differ-
ent days (but using the same S
35
RNA probes). Over 20 h incubation usually gener-
ates high background. Although general signal intensity may be less, 6-9 h of
incubation may generate a better S/N ratio.
