Samples collecting

The faecal samples of a nut size will be collected directly into a clean, wide-mouth
container and refrigerate at 4 C (do not use ice) until they were sent urgently to the
Endocrine er!ice at "ospital #ni!ersitario $irgen de la $ictoria %alaga& The samples
must be recei!ed before than ' or ( hours from their collection&
The samples )ept at 4 * C must be processed ( to obtain +,- or +.,) within (4 hours of
collection or frozen at -/0C until their use&
Extraction of bacterial RNA and DNA from faecal samples
1or .,+ e2traction, frozen faecal samples will be diluted '3'0 (w4!ol) in a '3' mi2ture of
.,+se-free water and .,+5ater (+mbion), thawed at 4*C, homogenized and centrifuged
at (00 g for 6 minutes& The supernatant will be transferred to a new tube and centrifuged at
'4&000 g for 6 minutes, and the pellet will be resuspended in '&( ml of a '3' mi2ture of
.,+se-free water and .,+5ater& The suspension will be transferred to a ( ml Eppendorf
tube containing appro2imately 600 mg of zirconia-silica beads (7iospec 8roducts 9nc&,
7artles!ille, :;, #+)& + <0 =l >uantity of '0? sodium dodecyl sulphate will be added,
and the bacteria will be lysed by sha)ing for 4 minutes on a bead beater (%i2er %ill %%
<00, .etch, "aan, @ermany) at <0 "z& +fter centrifugation at '000 g for (0s, the
supernatant will be transferred to a new tube and )ept at -(0*C until .,+ isolation by use
of Aiagen .,easy columns (Aiagen) according to the manufacturers instructions& .esidual
-,+ will be remo!ed by treatment with -,+-freeB (+mbion)& .,+ integrity will be
assessed by electrophoresis of each sample in a '&( agarose gel in which 'C and (< .,+
bands will be obser!ed&
Extraction of bacterial DNA from faecal samples
1or -,+ e2traction, frozen faecal samples will be diluted '3'0 (w4!ol) in phosphate-
buffered saline and thawed at 4*C& -,+ will be e2tracted from ( ml of the '0
-'
dilution &
The samples will be !orte2ed thoroughly and then centrifuged for ( min at (00 D g in a
microcentrifuge& The supernatant will be transferred to a new microcentrifuge tube and
centrifuged at '(,000 D g for 6 min& The supernatant will be discarded, and the pellet
resuspended in 6E0 =l of TE ('0 m% Tris-"Cl, ' m% E-T+, p" /&0)& The suspension will
be transferred to (-ml screw-cap tubes to which <60 to 400 mg of '00-=m zirconia-silica
beads (7iospec 8roducts 9nc&, 7artles!ille, :)la&) and <0 =l of '0? sodium dodecyl sulfate
will be added, and the bacterial cells will be lysed by being sha)en for 4 min on a minibead
beater (7iospec 8roducts 9nc&) on high speed& +fter a brief spin in a microcentrifuge, the
samples will be transferred to a microcentrifuge tube and the -,+ will be purified using
the A9+amp -,+ tool %ini ;it (Aiagen, "ilden, @ermany) and stored in <0 =l
autocla!ed water at -(0*C until use&
Analysis of faecal microbiota by DGGE
The $(-$< region of the 'C r.,+ genes (positions <<F to 6<F in the Escherichia coli
gene) of bacteria in the gut samples will be amplified by primers "-+'-@C (6G-C@C CC@
@@@ C@C @CC CC@ @@C @@@ @C@ @@@ @C+ C@@ @@@ @+C TCC T+C @@@
+@@ C+@ C+@ T-<GH the @C clamp is in boldface) and "-+( (6G-@T+ TT+ CC@ C@@
CT@ CT@ @C+ C-<G)&
DNA
+li>uots ('0 =l) of purified -,+ will be amplified by real-time 8C. ((0 ul final !olume)
(+pplied 7iosystems) using 1ast I7. @reen %aster %i2 and (00n% of each of the
uni!ersal primers "-+'-@C4"-+( with the following amplification program3 initial
denaturation was at F6* for (0 s, amplification was carried out using 40 cycles including
denaturation at F6*C for < s, annealing at C0*C for <0 s and e2tension at E(*C for '6 s&
RNA
+li>uots ('0 =l) of purified .,+ will be amplified using the "ight Capacity c-,+ re!erse
transcription )it (+pplied 7iosystems) according to the manufacturers instructions )it&
.e!erse transcription 8C. ((0 ul) will be run with the following amplification program3
one cycle consisting of C0 minutes at <E*C (re!erse transcription) and 6 minutes at F6*C,
followed by amplification of c-,+ ((0 ng4(0ul reaction) using the 1ast I7. @reen
%aster %i2 (+pplied 7iosystems) and (00n% of each of the uni!ersal primers "-+'-
@C4"-+( with the following amplification program3 3 initial denaturation was at F6* for
(0 s, amplification was carried out using 40 cycles including denaturation at F6*C for < s,
annealing at C0*C for <0 s and e2tension at E(*C for '6 s
PCR-DGGE
1ecal samples from each subJect will be e2amined by determining

8C.--@@E profiles&
Electrophoresis wil be performed with a -Code B #ni!ersal %utation -etection ystem
instrument (7io-.ad)& C? polyacrylamide gels will be prepared and

electrophoresed with
'D T+E buffer prepared from 60D T+E buffer

(( % Tris base, ' % glacial acetic acid,
60 m% E-T+)& The denaturing

gradient will be formed by using two C? acrylamide
(acrylamide4bisacrylamide

ratio, <E&63') stoc) solutions (7io-.ad)& The gels contained a
<0 to 66? gradient of urea and formamide that increase in the

direction of electrophoresis&
The '00? denaturant solution contain 40? formamide and E % urea& '< =l 8C. product
will be mi2ed with < =l loading dye before loading& Electrophoretic runs will be in a Tris-
acetate-E-T+ buffer (T+E '2) (40 mmol4l Tris, (0 mmol4l acetic acid, and ' mmol4l
E-T+, p" E&4) at '<0 $ and C0*C for 4&6 h& Electrophoresis

will be stopped when a 2ylene
cyanol dye mar)er reached the bottom

of a gel& @els will be stained with I7. afe '2
(9n!itrogen) for <0 min, rinsed with deionized water, and !iewed by #$ transillumination&
-enaturing gradient gel electrophoresis (-@@E) profiles will be compared by determining
the -ice similarity coefficient and using the 7ionumerics software pac)age (!ersion 4&0',
+pplied %aths) at a sensiti!ity of 'K(?&
Excision, cloning and seqencing of selected bands from DGGE gels
7ands of specific interest will be e2cised from -@@E gels with a sterile razor, placed in 40
=l sterile water, and incubated at 4*C for diffusion of -,+ into the water& << =l of the
sterile water (containing the -,+) will treated with ' nuclease& The ' nuclease treated
-,+ will be used in a 8C. with "-+'4( primers without @C-clamp (4 min at F4*C, (0
cycles consisting of <0 sec at F4*C, <0 sec at 6C*C, and ' min at C/*C, and finally E min at
C/*C)& ubse>uently the 8C. products will be directly cloned into pC.
L
4-T:8:
(9n!itrogen, #;) according to the manufacturerMs instructions& 8lasmid -,+ will be
isolated from the cells using the Aiagen %ini pin 8rep )it (A9+@E,, @ermany), and
subJected to 8C. ("-+'4(-@C) as earlier described& The 8C. product will be run on a
-@@E gel to chec) the purity and confirm the melting beha!ior of the e2cised band& The
inserts will be se>uenced using primers T< and TE& ,ucleotide se>uence data were
analyzed& The obtained se>uences will be compared to )nown se>uences in the @en7an)
-,+ database by using the

75+T algorithm&
Reasons for t!e se of DNA or RNA-DGGE
ince the culti!able part of the faecal microbiota probably constitutes only (0K60? of the
gut microbes it is important to e2plore effects on this comple2 ecosystem by use of
molecular fingerprinting methods allowing representation of the non-culti!able bacterial
species& +n e2ample of such a fingerprinting method is -enaturing @radient @el
Electrophoresis (-@@E) of 8C.-amplified 'C r.,+ genes, which ha!e pro!ed !ery
useful for analysis of faecal bacteria & Nhile the -@@E profiles based on amplified r.,+
genes (-,+--@@E) pro!ides a fingerprint of the composition of the in!estigated
community, they do not necessarily reflect metabolic acti!ities, and could e!en originate
from dormant, lysed or dead cells& The number of ribosomes in pro)aryotic cells is
correlated to growth rate, and profiles based on amplified ribosomal .,+ se>uences
(.,+--@@E) may better reflect the metabolically acti!e bacterial community& 9ndeed, a
recent study showed that alterations of bacterial community profiles after ingestion of
prebiotic oligosaccharides by human subJects were only detected in -@@E profiles
generated from bacterial r.,+&