 | Box 1
Nature Medicine
4, 1329 - 1333 (1998)
doi:10.1038/3327
Real-time quantitative RT−PCR after laser-assisted cell picking
Ludger Fink, Werner Seeger, Leander Ermert, Jörg Hänze, Ulrich Stahl, Friedrich Grimminger, Wolfgang Kummer
& Rainer M. Bohle | | | 
| Materials and methods
Lung isolation and perfusion. Lungs from male CD-rats (Sprague Dawley,
350−400 g; Charles River, Sulzfeld, Germany) were isolated, ventilated
and ex-vivo-perfused with Krebs-Henseleit buffer as previously described14,
15. Perfusion pressure, ventilation pressure and weight of isolated
organ were continuously registered.
Lungs selected for the study
had a homogeneous white appearance without signs of hemostasis or edema formation;
had pulmonary artery and ventilation pressures in the normal range; and were
isogravimetric during a steady-state period of 30 min.
After the
steady-state period, 75  g LPS and 1000 U IFN- in a total volume
of 5 ml were aerosolized into the afferent limb of the ventilator circuit
with an ultrasound vaporizer (Portasonic; De Vilbiss, Langen, Germany) for
10 min. For high-challenge lung, an additional stretching maneuver was performed
by increasing the end-expiratory pressure to 5 cm H2O (normal is
3 cm H2O) and repetitive doubling of tidal volume. Afterwards lungs
were perfused and ventilated under standard conditions for 6 h. Control lung
did not undergo these procedures. After termination of perfusion, the left
lung was lavaged (see below) while the right lung was instilled with TissueTek
via a cannula and snap frozen in liquid nitrogen.
Bronchoalveolar
lavage. Aliquots (3 ml) of saline were instilled into the left lung and
immediately reaspirated (total volume 18 ml). Cells were centrifuged at 1500
r.p.m., washed twice, and subsequently counted in a hemocytometer chamber.
Viability was assessed by trypan blue exclusion and differential counting
was performed (>97% alveolar macrophages, in all experiments). For direct
performance of mRNA extraction, aliquots of lavaged alveolar macrophages (25,000−45,000
cells)were lysed in 250 l homogenization buffer/2.5 l  -mercaptoethanol
of the MPG Guanidine Direct mRNA Purification Kit (CPG, Lincoln Park, New
Jerey). For in vitro stimulation, alveolar macrophages were distributed
to plastic Petri dishes and suspended in RPMI 1640 (Life Technologies, Paisley,
Scotland) supplemented with 2% rat serum. Alveolar macrophages were allowed
to adhere for 2 h at 37 °C and 5% CO2.
In vitro
stimulation. After 2 h adherence, medium was exchanged with a mixture
of 10 g/ml LPS and 1000 U/ml IFN-. Alveolar macrophages were then
incubated for 4, 6, 8, 10, and 14 h. For harvesting cells, medium was removed
and alveolar macrophages were lysed in 250 l homogenization buffer/2.5 l -mercaptoethanol.
Cell lysate was transferred into a 1.5 ml reaction tube and snap frozen in
liquid nitrogen for further RNA extraction.
mRNA extraction.
mRNA extraction was carried out using the MPG Guanidine Direct mRNA Purification
Kit based on magnetic separation of mRNA that is caught by attachment to oligo-dT
fragments. According to the manufacturer's protocol, oligo-dT fragments were
bound to streptavidin that is covalently coupled to the surface of supermagnetic
glass particles. For each sample, 150 g MPG−streptavidin were linked
to 1.5 l biotinylated oligo-dT. Isolated mRNA was finally solved in 20 l
DEPC-treated H2O.
Cytospin sections, tissue sections
and staining. For cytospin sections, one aliquot of lavaged alveolar macrophages
was washed twice. After assessing viability by trypan blue exclusion and differential
counting, cells were centrifuged in a 200-l reaction tube at 1500 r.p.m.
for 5 min, overlaid with TissueTek and snap frozen in liquid nitrogen. Next,
5-m sections of tube and the TissueTek-embedded alveolar macrophages were
prepared in a cryotome, mounted on glass slides (0.17-mm thickness), stained
with hematoxylin for 45 s, rinsed twice in DEPC-water and subsequently immersed
in 70%, 90%, and 100% ethanol. From tissue blocks of the TissueTek-fixed right
lung, 5-m cryosections were prepared in an identical manner.
Laser-assisted cell picking. The UV-laser microbeam (P.A.L.M., Wolfratshausen,
Germany) used for microdissection consists of a nitrogen laser of high-beam
precision (wavelength 337 nm), which is coupled to an inverted microscope
(Axiovert 135; Zeiss, Jena, Germany) via the epifluorescence illumination
path. Microscope stage and micromanipulator are digitally controlled and moved
by computer mouse. After selecting cells of interest, adjacent cells were
photolysed by laser beam (Fig. 3). A sterile needle
linked to the micromanipulator served for picking the selected cell(s) via
adhesive forces, with direct transfer into a reaction tube.
RNA processing from picked cells. Needles with adherent cell(s) were transferred
into a reaction tube containing 10 l of first-strand buffer (FSB). FSB
was prepared as described previously16 with slight modifications:
4% RNase inhibitor (Perkin Elmer, Überlingen, Germany) was added to 52 mM
Tris-HCl, pH 8.3, 78 mM KCl and 3.1 mM MgCl2. Nonionic detergent
was omitted. FSB and picked cells were incubated on ice for 5 min. Afterwards
specimens were snap-frozen in liquid nitrogen.
cDNA synthesis.
cDNA synthesis was done using products purchased from Perkin Elmer (10
 PCR-buffer II, MgCl2, random hexamers, RNase inhibitor, MULV
reverse transcriptase) and Eurobio (dNTPs; Raunheim, Germany). Tubes with
picked cells as well as 10 l of H2O-diluted mRNA from lavaged
alveolar macrophages were heated to 70 °C for 10 min and then cooled on ice
for 5 min. For cDNA synthesis of extracted mRNA, 4 l MgCl2
(5 mM), 2 l 10 PCR-buffer II, 1 l dNTP (10 mM each), 1 l random
hexamers (50 M), 0.5 l (10 U) RNase inhibitor and 1 l (50 U) MULV
reverse transcriptase were added to a total volume of 19.5 l. For cDNA
synthesis from picked alveolar macrophages, 4 l H2O, 1 l
dNTP (10 mM each), 1 l random hexamers (50 M), 0.5 l (10 U) RNase
inhibitor and 1 l (50 U) MULV reverse transcriptase were added to a total
volume of 17.5 l. Samples were incubated at 20 °C for 10 min and 43 °C
for 60 min. Reactions were stopped by heating to 99 °C for 5 min.
Real-time PCR. Cleavage of the sequence-specific probe by nuclease activity
releases the reporter dye resulting in an emission increase of respective
wavelength. PE ABI's Prism 7700 Sequence Detection System monitors emission
intensity continuously. The signal is normalized to an internal reference
( Rn) and the software sets the threshold cycle Ct, when Rn
becomes equal to ten standard deviations of the baseline. Ct is
used for quantitation of the input target number. For relative quantitation
as used here, comparative Ct method normalizes the number of target
gene copies to an endogenous reference called calibrator, for example, a suitable
housekeeping gene. Based on the exponential amplification of target gene,
as well as calibrator, the amount of amplified molecules at the threshold
cycle is given by (also described in ref. 11):
Xt: Number of molecules
at threshold cycle; X0: Initial number of molecules; Ex:
Efficiency of target amplification; ct: Threshold cycle for amplification;
and Kx: Constant.
Ratio of target gene copies (T) to
standard gene copies (R, calibrator reference) at threshold cycle normalizes
target gene expression for further comparison:
Assuming
same efficiency of target and reference gives the following expression:
and
After
cDNA synthesis, each sample of picked macrophages was divided for target gene
and standard gene analysis into two aliquots of 8 l. Incase of
separated mRNA, 1.5 l of cDNA each were applied. The TaqMan PCR Reagent
Kit (Perkin Elmer) was used according to the manufacturer's protocol with
slight modifications: dUTP was replaced by dTTP at the same concentration,
and incubation with AmpErase was omitted. MgCl2 concentration at
4 mM and 0.5 l (2.5 U) of AmpliTaq Gold Polymerase was tested to be optimal
in pilot experiments. Oligonucleotide primers (Table 1)
were added to a final concentration of 300 nM each and hybridization probe
(Table 1) to a final concentration of 200 nM in a
volume of 50 l. Oligonucleotides were synthesized by PE ABI (Weiterstadt,
Germany). Cycling conditions were modified to 94 °C for 2.45 min,
followed by 60 cycles of 94 °C for 45 s, 62 °C for 45 s and 73 °C for 45 s.
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