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Life science unlimited
Manual
!!"#$%#&' (")&$*+&, -
#"./ 01- 2!&*234&567,89:;<<7=7<><??<
This documentation describes the state at the time of
publishing. It needs not necessarily agree with future versions.
Subject to change!
© Copyright 2009, Analytik Jena AG, AJ Innuscreen GmbH
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Contents
1 Safety precautions ........................................................................... 3
2 Storage conditions ........................................................................... 3
3 Function testing and technical assistance.................................... 3
4 Product use and warranty ............................................................... 3
5 Kit components ................................................................................ 4
6 Recommended steps before starting............................................. 5
7 Components not included in the kit ............................................... 5
8 General notes and safety recommendations on handling RNA.. 6
9 General procedure for viral RNA extraction.................................. 7
10 Product specifications..................................................................... 7
&
11 Protocol 1: Isolation of viral RNA from serum, plasma, cell
culture supernatants or other cell-free body fluids........................ 8
12 Protocol 2: Isolation of viral RNA from tissue samples or
biopsies (max. 20 mg)..................................................................... 10
13 Protocol 3: Extraction of viral RNA from paraffin embedded
tissue sample .................................................................................. 12
14 Protocol 4: Isolation of viral RNA from swabs ............................. 14
15 Protocol 5: Isolation of viral RNA from cell cultures (max. 5 x 106
cells)................................................................................................. 16
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16 Troubleshooting ............................................................................. 18
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1 Safety precautions
+//& H"=& 01(=& 1!H& 1--=!- 2!& )I2"/H& .=& =J=(0 )=H& !& I1!H/ !K& -I=& L1-=( 1/)& 1!H&(=1K=!-)&02!-1 !=H& !&-I=&M -3&+/N1F)&N=1(&K/2O=)&NI /=&I1!H/ !K&-I=)=&(=1K=!-)&1!H&1O2 H&1!F&)M !&02!-10-P& A!&01)=&2E&02!-10-Q& E/")I&=F=)&2(&)M !&N -I&1& /1(K=&1L2"!-&2E&N1-=(& LL=H 1-=/F3&
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2 Storage conditions
RI=& !!"#$%#&' (")&$*+&, -&)I2"/H&.=&)-2(=H&H(FQ&1-& (22L&-=LG=(1-"(=&SD:T&CU&V@W&1!H& )&)-1./=&E2(&1-&/=1)-&DC&L2!-I)&"!H=(&-I=)=&02!H - 2!)3&6=E2(=&=O=(F&")=& L1M=& )"(=& -I1-& 1//& 02LG2!=!-)& I1O=& (22L& -=LG=(1-"(=3& AE& -I=(=& 1(=& 1!F&G(=0 G -1-=)& N -I !& -I=& G(2O H=H& )2/"- 2!)& )2/O=& -I=)=& G(=0 G -1-=)& .F& 01(=E"/&N1(L !K3&
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3 Function testing and technical assistance
RI=&+!1/F- M&X=!1&+Y&K"1(1!-==)&-I=&02((=0-&E"!0- 2!&2E&-I=&M -&E2(&1GG/ 01- 2!)&1)&H=)0( .=H& !&-I=&L1!"1/3&RI=&02LG2!=!-)&2E&=10I& !!"#$%#&' (")&$*+&, -N=(=&-=)-=H&.F& )2/1- 2!&2E&-I=&O (1/&$*+&1!H&)".)=Z"=!-&-1(K=-91LG/ E 01- 2!3&
[=& (=)=(O=& -I=& ( KI-& -2& 0I1!K=& 2(& L2H EF& 2"(& G(2H"0-)& -2& =!I1!0=& -I=(=&G=(E2(L1!0=&1!H&H=) K!3&AE&F2"&I1O=&1!F&Z"=)- 2!)&2(&G(2./=L)&(=K1(H !K&1!F&1)G=0-)& 2E& -I=& !!"#$%#& ' (")& $*+& , -& 2(& 2-I=(& +!1/F- M& X=!1& +Y& G(2H"0-)Q&G/=1)=&H2&!2-&I=) -1-=&-2&02!-10-&")3&\2(&-=0I! 01/&)"GG2(-&2(&E"(-I=(& !E2(L1- 2!& !&Y=(L1!F&G/=1)=&H 1/&]:>&?^&:D&B&;;&>:&<<3&\2(&2-I=(&02"!-( =)&G/=1)=&02!-10-&F2"(&/201/&H )-( ."-2(3&
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4 Product use and warranty
RI=&")=(& )&(=)G2!) ./=&-2&O1/ H1-=&-I=&G=(E2(L1!0=&2E&-I=&+!1/F- M&X=!1&+Y&M -)&E2(&1!F&G1(- 0"/1(&")=Q&) !0=&-I=&G=(E2(L1!0=&0I1(10-=( )- 0)&2E&2"(&M -)&I1O=&!2-&.==!&O1/ H1-=H&E2(&1!F&)G=0 E 0&1GG/ 01- 2!3&+!1/F- M&X=!1&+Y&M -)&L1F&.=&")=H& !& 0/ ! 01/& H 1K!2)- 0& /1.2(1-2(F& )F)-=L)& 1E-=(& -I=& /1.2(1-2(F& I1)& O1/ H1-=H& -I=&02LG/=-=&H 1K!2)- 0&)F)-=L&1)&(=Z" (=H&.F&@_A+`&aa&(=K"/1- 2!)& !& -I=&b383&2(&=Z" O1/=!-)& !&2-I=(&02"!-( =)3&
+//& G(2H"0-)& )2/H& .F& -I=& +!1/F- M& X=!1& +Y& 1(=& )".c=0-=H& -2& =J-=!) O=& Z"1/ -F&02!-(2/& G(20=H"(=)& 1!H& 1(=& N1((1!-=H& -2& G=(E2(L& 1)& H=)0( .=H& NI=!& ")=H&02((=0-/F3&+!F&G(2./=L)&)I2"/H&.=&(=G2(-=H& LL=H 1-=/F3&
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Note
\2(&(=)=1(0I&")=&2!/FP&
, -&02LG2!=!-)&
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5 Kit components
&
Important
+//&02LG2!=!-)&1(=&)-2(=H&1-&(22L&-=LG=(1-"(=3&
10 extractions 50 extractions 250 extractions
Lysis Solution RL ^&L/& CU&L/& DC<&L/&
Binding Solution RBS
a&L/& ?<&L/& D^<&L/&
Washing Solution HS
?&L/&SE !1/&O2/"L=&^&L/W&
DU&L/&SE !1/&O2/"L=&?<&L/W&
;<&L/&&SE !1/&O2/"L=&D:<&L/W&
Washing Solution LS
C&L/&SE !1/&O2/"L=&D<&L/W&
a&L/&SE !1/&O2/"L=&:<&L/W&
?^&L/&SE !1/&O2/"L=&Da<&L/W&
RNAse-free water C&L/& ^&L/& ?<&L/&
Spin Filter D<& U<& U&J&U<&
Receiver Tubes SC3<&L/W
U<& U&J&U<& CU&J&U<&
Elution Tubes SD3U&L/W
D<& U<& U&J&U<&
Manual D& D& D&
Initial steps !+HH&?&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I9 !K&82/"- 2!&58Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2-9-/=&1/N1F)&E (L/F&
0/2)=HP&
!+HH&a&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I9 !K&82/"- 2!&_8Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2-9-/=&1/N1F)&E (L/F&
0/2)=HP&
!+HH&DU&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I9 !K&82/"- 2!&58Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2-9-/=&1/N1F)&E (L/F&
0/2)=HP&
!+HH&?C&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I9 !K&82/"- 2!&_8Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2-9-/=&1/N1F)&E (L/F&
0/2)=HP&
!+HH&;<&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I !K&82/"- 2!&58Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2--/=&1/9
N1F)&E (L/F&0/2)=HP&
!+HH&D::&L/&2E&>^9>>3a&d&=-I1!2/&-2&-I=&.2--/=&[1)I !K&82/"- 2!&_8Q&L J&-I2(2"KI/F&1!H&M==G&-I=&.2--/=&1/9
N1F)&E (L/F&0/2)=HP&
& $=02LL=!H=H&)-=G)&.=E2(=&)-1(- !K&
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6 Recommended steps before starting
!%!)"(=& -I1-& -I=&[1)I !K&82/"- 2!&58&1!H&[1)I !K&82/"- 2!&_8&I1O=&.==!&G(=G1(=H&1002(H !K&-2&-I=& !)-("0- 2!&Se&f, -&02LG2!=!-)f&G3&:W&
!@=!-( E"K1- 2!&)-=G)&)I2"/H&.=&01(( =H&2"-&1-&(22L&-=LG=(1-"(=3&
!+O2 H&E(==g !K&1!H&-I1N !K&2E&)-1(- !K&L1-=( 1/3&
7 Components not included in the kit
!hF/=!=&2(&20-1!i&2G- 2!1/&
! D3U&L/&(=10- 2!&-".=)&
! C&L/&(=10- 2!&-".=)i&2G- 2!1/&
! >^&T&>>3a&d&=-I1!2/&
! HH5Cj&&
&
8 Addition of Carrier RNA (opional)
!jG- 2!1//FQ&E2(&-I=& )2/1- 2!&2E&O (1/&$*+&E(2L&)1LG/=)&02!-1 ! !K&=J-(=L=/F&/2N& 1L2"!-)& 2E& I2)-& !"0/= 0& 10 H)& S=3K3& )=("LQ& G/1)L1& 1!H& 2-I=(& 0=//9E(==&)-1(- !K& L1-=( 1/)W& 1!H& 02!-1 ! !K& 1!& =JG=0-=H& O=(F& /2N& 1L2"!-& 2E& O (1/&!"0/= 0&10 H)& -&02"/H&.=&I=/GE"/&-2&1HH&@1(( =(&$*+&-2&-I=&. !H !K&)-=G&S1E-=(&/F) )&W3&
! A-& )& (=02LL=!H=H& -2& ")=& @1(( =(& $*+& S=3K3& #2/FS+W& $*+i& $20I=&k 1K!2)- 0)i&*23D<a^C^W3&
! k ))2/O=&-I=&@1(( =(&$*+& !&$*1)=9E(==&N1-=(&-2&2.-1 !&1&)2/"- 2!&2E&D&lKBl/3&k O H=& !-2&1/ Z"2-)&1!H&)-2(=&1-&TC<V@3&
! k2&!2-&E(==g=&1!H&-I1N&-I=&1/ Z"2-)&L2(=&-I1!&?&- L=)3&
! A-& )&(=02LL=!H&-2&1HH&U&l/&2E&-I=&@1(( =(&$*+&SU&lKW&G=(&)1LG/=&
&
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Y=!=(1/&!2-=)&1!H&)1E=-F&(=02LL=!H1- 2!)&2!&I1!H/ !K&$*+&
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9 General notes and safety recommendations on handling RNA
$*+& )& E1(& /=))& )-1./=& -I1!& k*+3& A-& )& O=(F& )=!) - O=& -2& H=K(1H1- 2!& .F&=!H2K=!2")& $*1)=)& !& -I=& . 2/2K 01/& L1-=( 1/& 1!H& =J2K=!2")& $*1)=)& NI 0I&1(=& G=(L1!=!-/F& G(=)=!-& =O=(FNI=(=& !& -I=& /1.3& R2& 10I =O=& )1- )E10-2(F&Z"1/ -1- O=& 1!H& Z"1!- -1- O=& (=)"/-)& !& $*+& G(=G1(1- 2!)Q& 02!-1L !1- 2!)& N -I&=J2K=!2")& $*1)=)& I1O=& -2& .=&(=H"0=H& -2& 1& L ! L"L& !& 1002(H1!0=& N -I& -I=&E2//2N !K&(=02LL=!H1- 2!)4&
!+/N1F)&N=1(&/1-=J&2(&O !F/&K/2O=)&NI /=&I1!H/ !K&(=1K=!-)&1!H&$*+&)1LG/=)&-2& G(=O=!-& $*1)=& 02!-1L !1- 2!)& E(2L& )"(E10=& 2E& -I=& )M !& 2(& E(2L& H")-F&/1.2(1-2(F&=Z" GL=!-3&
!@I1!K=&K/2O=)&E(=Z"=!-/F&1!H&M==G&-".=)&0/2)=H3&
!,==G& )2/1-=H&$*+&2!& 0=3&&
!$=H"0=&G(=G1(1- 2!&- L=&1)&L"0I&1)&G2)) ./=3&
!b)=& 2!/F& )-=( /=Q& H )G2)1./=& G2/FG(2GF/=!=& -".=)& -I(2"KI2"-& -I=& G(20=H"(=&S-I=)=&-".=)&1(=&K=!=(1//F&$*1)=9E(==3W&
!*2!9H )G2)1./=&G/1)- 0&N1(=&)I2"/H&.=&-(=1-=H&.=E2(=&")=&-2&=!)"(=&-I1-& -& )&$*1)=9E(==3& #/1)- 0& N1(=& )I2"/H& .=& -I2(2"KI/F& ( !)=H& N -I& <3D& m& *1j5Q&D&Lm& %kR+& E2//2N=H& .F& $*1)=9E(==& N1-=(3& n2"& 01!& 1/)2& -1M=& 0I/2(2E2(L9(=) )-1!-&G/1)- 0&N1(=&( !)=H&N -I&0I/2(2E2(L&-2& !10- O1-=&$*1)=)3&
!+//&K/1))N1(=&)I2"/H&.=& -(=1-=H&.=E2(=&")=& -2&=!)"(=& -I1-& -& )&$*1)=9E(==3&Y/1))N1(=& )I2"/H& .=& 0/=1!=H& N -I& H=-=(K=!-Q& -I2(2"KI/F& ( !)=H& 1!H& 2O=!&.1M=H&1-&C:<&V@&E2(&E2"(&2(&L2(=&I2"()&.=E2(=&")=3&+"-20/1O !K&1/2!=&N //&!2-& !10- O1-=& L1!F& $*1)=)& 02LG/=-=/F3& jO=!& .1M !K& !10- O1-=)& $*1)=)& 1!H&=!)"(=)& -I1-&!2&2-I=(&!"0/= 0&10 H)&S)"0I&1)&G/1)L H&k*+W&1(=&G(=)=!-&2!&-I=& )"(E10=& 2E& -I=& K/1))N1(=3& n2"& 01!& 1/)2& 0/=1!& K/1))N1(=& N -I& <3D&d&k%#@&SH =-IF/&GF(201(.2!1-=W3&RI=&K/1))N1(=&I1)&-2&.=& LL=()=H& !&<3D&d&k%#@& )2/"- 2!& E2(& DC& I2"()& 1-& ?;&V@& 1!H& -I=!& -& I1)& -2& .=& 1"-20/1O=H& 2(&I=1-=H&-2&D<<&V@&E2(&DU&L !&-2&(=L2O=&(=) H"1/&k%#@3&
!%/=0-(2GI2(=) )&-1!M)&)I2"/H&.=&0/=1!=H&N -I&H=-=(K=!-&)2/"- 2!&S=3K3&<3U&d&8k8WQ& -I2(2"KI/F& ( !)=H& N -I& $*1)=9E(==& N1-=(Q& ( !)=H& N -I& =-I1!2/& 1!H&E !1//F&1//2N=H&-2&H(F3&
!+//&."EE=()&I1O=&-2&.=&G(=G1(=H&N -I&k%#@9-(=1-=H&$*1)=9E(==&HH5Cj3&
!+O2 H&I1!H/ !K&.10-=( 1/&0"/-"(=)Q&0=//&0"/-"(=)&2(&2-I=(&. 2/2K 01/&)2"(0=)&2E&$*1)=)& !&-I=&)1L=&/1.&NI=(=&-I=&$*+&G"( E 01- 2!&N //&.=&G=(E2(L=H3&
!k2& !2-& ")=& =Z" GL=!-Q& K/1))N1(=& 1!H& G/1)- 0& N1(=& =LG/2F=H& E2(& 2-I=(&1GG/ 01- 2!)& NI 0I& L KI-& !-(2H"0=& $*1)=& 02!-1L !1- 2!)& !& -I=& $*+& )2/1- 2!3&
& Y=!=(1/&G(20=H"(=&E2(&O (1/&$*+&=J-(10- 2!&
&
!!"#$%#&' (")&$*+&, -& A))"=&<?BC<<>& ;&
10 General procedure for viral RNA extraction
&
Lysis of starting material
&
&
Binding of viral RNA on Spin Filter
&
&
&
&
Washing of the bound RNA
&
&
&
&
&Elution of RNA
&
&
11 Product specifications
&
1. Starting material:&
!8=("LQ& G/1)L1Q& 0=//& 0"/-"(=& )"G=(!1-1!-)& 1!H& 2-I=(& 0=//9E(==&.2HF&E/" H)&SDU<&l/W&
!R ))"=&)1LG/=)&1!H&. 2G) =)&SL1J3&C<&LKW&
!8N1.&)1LG/=)&
!#1(1EE !&=L.=HH=H&L1-=( 1/&
!@=//&0"/-"(=)&SL1J3&U&J&D<^W&
&
2. Time for isolation:&
+GG(2J L1-=/F&CU&L !"-=)&
&
3. Positive tested for:&
%3K3&5A'Q&5@'Q&6'k'Q&A!E/"=!g1&
#(2-202/&D4&A)2/1- 2!&2E&O (1/&$*+&E(2L&)=("LQ&G/1)L1Q&0=//&0"/-"(=&)"G=(!1-1!-)&2(&2-I=(&0=//9E(==&.2HF&E/" H)&
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a& A))"=&<?BC<<>& !!"#$%#&' (")&$*+&, -&
12 Protocol 1: Isolation of viral RNA from serum, plasma, cell culture supernatants or other cell-free body fluids
D3& # G=--=&450 µl Lysis Solution RL& !-2&1&D3U&L/&(=10- 2!&-".=&1!H&1HH&150 µl of the sampleQ&L J&O K2(2")/F&.F&G"/)=H&O2(-=J !K&E2(&D<&)=03&A!0".1-=&1-&(22L&-=LG=(1-"(=&E2(&DU&L !"-=)3&
Note:&[=&(=02LL=!H&-2&")=&1&)I1M !K&G/1-E2(L&E2(&1&02!- !"2")&)I1M !K&2E&-I=&)1LG/=3&+/-=(!1- O=/FQ&O2(-=J&-I=&)1LG/=&?9:&- L=)&H"( !K& !0".1- 2!3&*2&)I1M !K&N //&(=H"0=&-I=&/F) )&=EE 0 =!0F3&
+E-=(& /F) )& 0=!-( E"K=& -I=& D3U& L/& (=10- 2!& -".=& )I2(-/F& -2& (=L2O=&02!H=!)1-=&E(2L&-I=&/ H&2E&-I=&-".=3&&
C3& +HH&600 µl Binding Solution RBS& -2& -I=& /F)=H& )1LG/=Q& L J& .F&O2(-=J !K&2(&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
Note:& A-& )& LG2(-1!-& -I1-& -I=& )1LG/=& 1!H& -I=& 6 !H !K& 82/"- 2!& $68& 1(=&L J=H&O K2(2")/F&-2&K=-&1&I2L2K=!=2")&)2/"- 2!3&AE&-I=&=JG=0-=H&1L2"!-&2E&O (1/&$*+& )&O=(F&/2N&G/=1)=&1HH&U&l/&@1(( =(&$*+Q&1)&H=)0( .=H&1.2O=3&
?3& +GG/F&650 µl of the sample& -2&-I=&8G !&\ /-=(&/201-=H& !&1&C3<&L/&$=0= O=(& R".=3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K&
S"DC3<<<&(GLW&E2(&D&L !"-=3&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H&-I=&$=0= O=(&R".=&N -I&-I=&E /-(1-=3&
:3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& !=N& C3<& L/& $=0= O=(& R".=& 1!H& /21H&-I=& (=) H"1/& )1LG/=& 2!-2& -I=& 8G !& \ /-=(3& @/2)=& -I=& 01G& 1!H&
0=!-( E"K=&1-&D<3<<<&J&K&S"DC3<<<&(GLW&E2(&D&L !3&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&
0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
U3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&500 µl Washing Solution HSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K& S"DC3<<<& (GLW& E2(& D& L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
^3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&650 µl Washing Solution LSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K& S"DC3<<<& (GLW& E2(& D& L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
;3& @=!-( E"K=& 1-& L1J3& )G==H& E2(& C& L !"-=)& -2& (=L2O=& 1//& -(10=)& 2E&=-I1!2/3&k )01(H&-I=&C3<&L/&$=0= O=(&R".=3&
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RI=&$*+&01!&.=&=/"-=H& N -I&1& /2N=(&2(&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(& SH=G=!H)& 2!& -I=& =JG=0-=H& F =/H& 2E& $*+W3& %/"- 2!& N -I& /2N=(& O2/9"L=)& 2E& $*1)=9E(==& N1-=(& !0(=1)=)& -I=& E !1/& 02!0=!-(1- 2!& 2E& $*+3&8-2(=&-I=&=J-(10-=H&O (1/&$*+&1-&9C<&V@3&\2(&/2!K&- L=&)-2(1K=&G/10 !K&1-&9a<&V@& )&(=02LL=!H=H3&
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
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innuPREP Virus RNA Kit
Protocol 1: Isolation of viral RNA from cell-free body fluids
&$=02LL=!H=H&)-=G)&.=E2(=&)-1(- !K&
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"!D<3<<<&J&K&S"DC3<<<&(GLW4&D&L !&
"!_21H&(=) H"1/&)1LG/=&
"!D<3<<<&J&K&S"DC3<<<&(GLW4&D&L !&
& & & & && & & & &:3&& **jG- 2!1/4&+HH - 2!&2E&@1(( =(&$*+& & "!+HH&U&l/&SU&lKW&G=(&)1LG/=&
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
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Expression and further use permitted with indication of
source.
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Publisher: +!1/F- M&X=!1&+Y& & &
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
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innuPREP Virus RNA Kit
Protocol 2: Isolation of viral RNA from tissue samples or biopsies
&
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
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Expression and further use permitted with indication of
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Publisher: +!1/F- M&X=!1&+Y& & &
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
1/ 2
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innuPREP Virus RNA Kit
Protocol 3: Isolation of viral RNA from paraffin embedded tissue
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Expression and further use permitted with indication of
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© 2009 Analytik Jena AG
Publisher: +!1/F- M&X=!1&+Y& & &
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Short operation manual – innuPREP Virus RNA Kit Puplication No.: HB_KS-4700_e_090330
1/ 2
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innuPREP Virus RNA Kit
Protocol 4: Isolation of viral RNA from buccal swab
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source.
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Publisher: +!1/F- M&X=!1&+Y& & &
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13 Protocol 2: Isolation of viral RNA from tissue samples or biopsies (max. 20 mg)
D3& @"-&max. 20 mg of the sample& !-2&)L1//&G =0=)&1!H&G/10=& -I=&- ))"=& !&1&D3U&L/&(=10- 2!&-".=3&52L2K=! g=&-I=&)1LG/=&2(&K( !H&-I=& )1LG/=& L=0I1! 01//FQ& E& -& )& G2)) ./=3& +HH& 450 µl Lysis Solution RL& 1!H& L J& O K2(2")/F& .F& G"/)=H& O2(-=J !K& E2(& D<& )=03&A!0".1-=&1-&(22L&-=LG=(1-"(=&E2(&?<&T&^<&L !3&
Note:&[=&(=02LL=!H&-2&")=&1&)I1M !K&G/1-E2(L&E2(&1&02!- !"2")&)I1M !K&2E&
-I=&)1LG/=3&+/-=(!1- O=/FQ&O2(-=J&-I=&)1LG/=&U&- L=)&H"( !K& !0".1- 2!&E2(&D<&)=02!H)3&*2&)I1M !K&N //&(=H"0=&-I=&/F) )&=EE 0 =!0F3&
+E-=(& /F) )& 0=!-( E"K=& -I=& -".=& 1-& L1J3& )G==H& E2(& D& L !& -2& G=//=-&H2N!& "!/F)=H& L1-=( 1/3& R(1!)E=(& )"G=(!1-1!-& !-2& 1& !=N& D3U& L/&-".=3&
C3& +HH&450 µl Binding Solution RBS& -2& -I=& /F)=H& )1LG/=Q& L J& .F&O2(-=J !K&2(&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
Note:& A-& )& LG2(-1!-& -I1-& -I=& )1LG/=& 1!H& -I=& 6 !H !K& 82/"- 2!& $68& 1(=&L J=H&O K2(2")/F&-2&K=-&1&I2L2K=!=2")&)2/"- 2!3&
?3& +GG/F&650 µl of the sample&-2&-I=&8G !&\ /-=(&/201-=H& !&1&C3<&L/&$=0= O=(& R".=3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K&
S"DC3<<<&(GLW&E2(&D&L !3&&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
:3& k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& !=N& C3<& L/& $=0= O=(& R".=& 1!H& /21H& -I=& (=) H"1/& )1LG/=&2!-2& -I=& 8G !& \ /-=(3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K&
S"DC3<<<&(GLW&E2(&D&L !3&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&
0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
U3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&500 µl Washing Solution HSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K& S"DC3<<<& (GLW& E2(& D& L !3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3
^3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&650 µl Washing Solution LSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K& S"DC3<<<& (GLW& E2(& D& L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
& #(2-202/&C4&A)2/1- 2!&2E&O (1/&$*+&E(2L&- ))"=&)1LG/=)&2(&. 2G) =)&SL1J3&C<&LKW&
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^3<<<&J&K& S"a3<<<&(GLW& E2(& D& L !3& RN2& =/"- 2!& )-=G)& N -I& =Z"1/&O2/"L=)&2E&$*+)=9E(==&N1-=(&S=3K3&?<&l/&]&?<&l/W&L KI-& !0(=1)=&-I=&F =/H&2E&=J-(10-=H&O (1/&$*+3&
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Note
RI=&$*+&01!&.=&=/"-=H& N -I&1& /2N=(&2(&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(& SH=G=!H)& 2!& -I=& =JG=0-=H& F =/H& 2E& $*+W3& %/"- 2!& N -I& /2N=(& O2/9"L=)& 2E& $*1)=9E(==& N1-=(& !0(=1)=)& -I=& E !1/& 02!0=!-(1- 2!& 2E& $*+3&8-2(=&-I=&=J-(10-=H&O (1/&$*+&1-&9C<&V@3&\2(&/2!K&- L=&)-2(1K=&G/10 !K&1-&9a<&V@& )&(=02LL=!H=H3&
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14 Protocol 3: Extraction of viral RNA from paraffin embedded tissue sample
D3& #/10=& 1& G =0=& 2E& )-1(- !K&L1-=( 1/& !-2& 1& C3<&L/& (=10- 2!& -".=Q& 1HH&1 ml Octane or Xylene 1!H& O2(-=J& 01(=E"//F& -2& H ))2/O=& -I=&G1(1EE !3& \2//2N& -I=& H ))2/"- 2!& "!- /& -I=& - ))"=& )1LG/=& /22M)&-(1!)G1(=!-&SNI /=&G1(1EE !&(=L1 !)&NI -=W3&
C3& @=!-( E"K=&1-&L1J3&)G==H&E2(&U&L !&1-&(22L&-=LG=(1-"(=3&k )01(H&-I=&)"G=(!1-1!-&O=(F&01(=E"//F&.F&1)G (1- !K&N -I&1&G G=--=3&
Don’t&remove the pellet!&
Note:&RI )&)-=G&)I2"/H&.=&(=G=1-=H& E&1!F&G1(1EE !& )&)- //& !&-I=&)1LG/=3&
?3& +HH&1 ml ethanol&S>^T>>3a&dW&-2&-I=&G=//=-&1!H&O2(-=J&O K2(2")/F3&@=!-( E"K=& 1-& L1J3& )G==H& 1-& (22L& -=LG=(1-"(=& E2(& ?& L !& 1!H&(=L2O=&-I=&=-I1!2/&.F&G G=-- !K3&
Do not remove the pellet!
$=G=1-&-I=&N1)I !K&)-=G&N -I&=-I1!2/&2!0=&1K1 !3
:3& A!0".1-=& -I=&2G=!&-".=& 1-&?;& V@&E2(& D<TDU&L !& -2&=O1G2(1-=& -I=&(=) H"1/&=-I1!2/&02LG/=-=/F3&&
U3& +HH& 450 µl Lysis Solution RL3& R(1!)E=(& -I=& )1LG/=& S_F) )&82/"- 2!&$_&1!H&-I=&H=G1(1EE !=H&L1-=( 1/W& !-2&1&D3U&L/&(=10- 2!&-".=&1!H&L J&O K2(2")/F&.F&G"/)=H&O2(-=J !K&E2(&D<&)=03& A!0".1-=&1-&(22L&-=LG=(1-"(=&E2(&?<&L !3&
Note:&[=&(=02LL=!H&-2&")=&1&)I1M !K&G/1-E2(L&E2(&1&02!- !"2")&)I1M !K&2E&
-I=&)1LG/=3&+/-=(!1- O=/FQ&O2(-=J&-I=&)1LG/=&?T:&- L=)&H"( !K& !0".1- 2!3&*2&)I1M !K&N //&(=H"0=&-I=&/F) )&=EE 0 =!0F3&
+E-=(& /F) )& 0=!-( E"K=& -I=& -".=& 1-& L1J3& )G==H& E2(& D& L !& -2& G=//=-&H2N!& "!/F)=H& L1-=( 1/3& R(1!)E=(& )"G=(!1-1!-& !-2& 1& !=N& D3U& L/&-".=3&
^3& +HH&450 µl Binding Solution RBS& -2& -I=& /F)=H& )1LG/=Q& L J& .F&O2(-=J !K&2(&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
Note:& A-& )& LG2(-1!-& -I1-& -I=& )1LG/=& 1!H& -I=& 6 !H !K& 82/"- 2!& $68& 1(=&L J=H&O K2(2")/F&-2&K=-&1&I2L2K=!=2")&)2/"- 2!3&AE&-I=&=JG=0-=H&1L2"!-&2E&O (1/&k*+& )&O=(F&/2N&G/=1)=&1HH&U&l/&@1(( =(&$*+&1)&H=)0( .=H&1.2O=P&
;3& +GG/F&650 µl of the sample&-2&-I=&8G !&\ /-=(&/201-=H& !&1&C3<&L/&$=0= O=(& R".=3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K&
S"DC3<<<&(GLW&E2(&D&L !3&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&
0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
a3& k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& !=N& C3<& L/& $=0= O=(& R".=& 1!H& /21H& -I=& (=) H"1/& )1LG/=&
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Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
>3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&500 µl Washing Solution HSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
D<3jG=!&-I=&8G !&\ /-=(&1!H&1HH&650 µl Washing Solution LSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
DD3@=!-( E"K=& 1-& L1J3& )G==H& E2(& C& L !"-=)& -2& (=L2O=& 1//& -(10=)& 2E&=-I1!2/3&k )01(H&-I=&C3<&L/&$=0= O=(&R".=3&
DC3#/10=& -I=& 8G !& \ /-=(& !-2& 1& D3U& L/& %/"- 2!& R".=3& @1(=E"//F& 2G=!&-I=&01G&2E& -I=&8G !&\ /-=(&1!H& 1HH&60–100 µl RNase-free water3&A!0".1-=& 1-& (22L& -=LG=(1-"(=& E2(& C&L !3& @=!-( E"K=& 1-& ^3<<<&J&K&
S"a3<<<& (GLW& E2(&D&L !3&RN2&=/"- 2!&)-=G)& N -I&=Z"1/&O2/"L=)&2E&$*1)=9E(==& N1-=(& S=3K3&?<&l/&]&?<&l/W& L KI-& !0(=1)=& -I=& F =/H& 2E&=J-(10-=H&O (1/&$*+3&
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Note
RI=&$*+&01!&.=&=/"-=H& N -I&1& /2N=(&2(&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(& SH=G=!H)& 2!& -I=& =JG=0-=H& F =/H& 2E& $*+W3& %/"- 2!& N -I& /2N=(& O2/9"L=)& 2E& $*1)=9E(==& N1-=(& !0(=1)=)& -I=& E !1/& 02!0=!-(1- 2!& 2E& $*+3&8-2(=&-I=&=J-(10-=H&O (1/&$*+&1-&9C<&V@3&\2(&/2!K&- L=&)-2(1K=&G/10 !K&1-&&9a<&V@& )&(=02LL=!H=H3&
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15 Protocol 4: Isolation of viral RNA from swabs
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Important
R2&K=-&L1J L"L&F =/H&2E&$*+& -& )&=))=!- 1/&-2&/=1O=&-I=&)N1.&H"( !K&-I=&02LG/=-=&/F) )&- L=& !&-I=&D3U&L/&(=10- 2!&-".=3&A-& )&G2)) ./=&-2&0"-&-I=&)I1E-&2E&-I=&)N1.Q&)2&-I1-&F2"&01!&0/2)=&-I=&01G&2E&-I=&-".=3&RI=&(=L2O1/& 2E& -I=& )N1.& E(2L& -I=& -".=&1I=1H& 2E& - L=& N //& /=1H& -2& 1& H(19L1- 01//F&(=H"0=H&E !1/&F =/HP&
&
D3& #/10=& -I=& )N1.& !-2& 1& D3U& L/& (=10- 2!& -".=3& +HH& 450 µl Lysis Solution RL& 1!H& L J& O K2(2")/F& .F& G"/)=H& O2(-=J !K& E2(& D<& )=03&A!0".1-=&1-&(22L&-=LG=(1-"(=&E2(&DU&L !3&
Note:&[=&(=02LL=!H&-2&")=&1&)I1M !K&G/1-E2(L&E2(&1&02!- !"2")&)I1M !K&2E&
-I=&)1LG/=3&+/-=(!1- O=/FQ&O2(-=J&-I=&)1LG/=&?9:&- L=)&H"( !K& !0".1- 2!3&*2&)I1M !K&N //&(=H"0=&-I=&/F) )&=EE 0 =!0F3&
+E-=(& /F) )& 0=!-( E"K=& -I=& D3U& L/& (=10- 2!& -".=& )I2(-/F& -2& (=L2O=&02!H=!)1-=&E(2L&-I=&/ H&2E&-I=&-".=3&
C3& +HH&450 µl Binding Solution RBS& -2& -I=& /F)=H& )1LG/=Q& L J& .F&O2(-=J !K&2(&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
Note:& A-& )& LG2(-1!-& -I1-& -I=& )1LG/=& 1!H& -I=& 6 !H !K& 82/"- 2!& $68& 1(=&L J=H&O K2(2")/F&-2&K=-&1&I2L2K=!=2")&)2/"- 2!3&AE&-I=&=JG=0-=H&1L2"!-&2E&O (1/&k*+& )&O=(F&/2N&G/=1)=&1HH&U&l/&@1(( =(&$*+&1)&H=)0( .=H&1.2O=P&
?3& +GG/F&650 µl of the sample& -2&-I=&8G !&\ /-=(&/201-=H& !&1&C3<&L/&$=0= O=(& R".=3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K&
S"DC3<<<&(GLW&E2(&D&L !3&&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&
0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
:3& k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& !=N& C3<& L/& $=0= O=(& R".=& 1!H& /21H& -I=& (=) H"1/& )1LG/=&2!-2& -I=& 8G !& \ /-=(3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K&
S"DC3<<<&(GLW&E2(&D&L !3&&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
U3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&500 µl Washing Solution HSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&H )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
& #(2-202/&:4&A)2/1- 2!&2E&O (1/&$*+&E(2L&)N1.)&
&
!!"#$%#&' (")&$*+&, -& A))"=&<?BC<<>& DU&
^3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&650 µl Washing Solution LSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
;3& @=!-( E"K=& 1-& L1J3& )G==H& E2(& C& L !"-=)& -2& (=L2O=& 1//& -(10=)& 2E&=-I1!2/3&k )01(H&-I=&C3<&L/&$=0= O=(&R".=3&
a3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& D3U& L/& %/"- 2!& R".=3& @1(=E"//F& 2G=!&-I=& 01G& 2E& -I=& 8G !& \ /-=(& 1!H& 1HH&60-100 µl RNase-free water3&A!0".1-=& 1-& (22L& -=LG=(1-"(=& E2(& C&L !3& @=!-( E"K=& 1-& ^3<<<&J&K&
S"a3<<<&(GLW&E2(&D&L !"-=3&RN2&=/"- 2!&)-=G)&N -I&=Z"1/&O2/"L=)&2E& $*1)=9E(==& N1-=(& S=3K3&?<&l/&]&?<&l/W& L KI-& !0(=1)=& -I=& F =/H&2E&=J-(10-=H&O (1/&$*+3&
&
Note
RI=&$*+&01!&.=&=/"-=H& N -I&1& /2N=(&2(&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(& SH=G=!H)& 2!& -I=& =JG=0-=H& F =/H& 2E& $*+W3& %/"- 2!& N -I& /2N=(& O2/9"L=)& 2E& $*1)=9E(==& N1-=(& !0(=1)=)& -I=& E !1/& 02!0=!-(1- 2!& 2E& $*+3&8-2(=&-I=&=J-(10-=H&O (1/&$*+&1-&9C<&V@3&\2(&/2!K&- L=&)-2(1K=&G/10 !K&1-&9a<&V@& )&(=02LL=!H=H3&
&
#(2-202/&U4&A)2/1- 2!&2E&O (1/&$*+&E(2L&0=//&0"/-"(=)&SL1J3&U&J&D<^&0=//)W&
&
&
D^& A))"=&<?BC<<>& !!"#$%#&' (")&$*+&, -&
16 Protocol 5: Isolation of viral RNA from cell cultures (max. 5 x 106 cells)
D3& #=//=-&0=//)&.F&0=!-( E"K1- 2!&E2(&D<&L !&1-&U3<<<&J&K&S";3U<<&(GLW3&k )01(H&)"G=(!1-1!-3&
C3& +HH&450 µl Lysis Solution RL&1!H&(=9)")G=!H&-I=&0=//&G=//=-&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
?3& R(1!)E=(&-I=&(=9)")G=!H=H&0=//)& !-2&1!&D3U&L/&(=10- 2!& -".=&1!H&L J& O K2(2")/F& .F& G"/)=H& O2(-=J !K& E2(& D<& )=03& A!0".1-=& 1-& (22L&-=LG=(1-"(=&E2(&DU&L !3&
Note:&[=&(=02LL=!H&-2&")=&1&)I1M !K&G/1-E2(L&E2(&1&02!- !"2")&)I1M !K&2E&-I=&)1LG/=3&+/-=(!1- O=/FQ&O2(-=J&-I=&)1LG/=&?T:&- L=)&H"( !K& !0".1- 2!3&*2&)I1M !K&N //&(=H"0=&-I=&/F) )&=EE 0 =!0F3&
+E-=(& /F) )& 0=!-( E"K=& -I=& D3U& L/& (=10- 2!& -".=& )I2(-/F& -2& (=L2O=&02!H=!)1-=&E(2L&-I=&/ H&2E&-I=&-".=3&
:3& +HH&450 µl Binding Solution RBS& -2& -I=& /F)=H& )1LG/=Q& L J& .F&O2(-=J !K&2(&.F&G G=-- !K&"G&1!H&H2N!&)=O=(1/&- L=)3&
Note:& A-& )& LG2(-1!-& -I1-& -I=& )1LG/=& 1!H& -I=& 6 !H !K& 82/"- 2!& $68& 1(=&
L J=H&O K2(2")/F&-2&K=-&1&I2L2K=!=2")&)2/"- 2!3&
U3& +GG/F&650 µl of the sample& -2&-I=&8G !&\ /-=(&/201-=H& !&1&C3<&L/&$=0= O=(& R".=3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K&
S"DC3<<<&(GLW&E2(&D&L !3&&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
^3& k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2& 1& !=N& C3<& L/& $=0= O=(& R".=& 1!H& /21H& -I=& (=) H"1/& )1LG/=&2!-2& -I=& 8G !& \ /-=(3& @/2)=& -I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<& J& K&
S"DC3<<<&(GLW&E2(&D&L !3&&
Note:& AE& -I=& )2/"- 2!& I1)& !2-& 02LG/=-=/F& G1))=H& -I(2"KI& -I=& 8G !& \ /-=(Q&0=!-( E"K=&1K1 !&1-&I KI=(&)G==H&2(&G(2/2!K&-I=&0=!-( E"K1- 2!&- L=3&
k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
;3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&500 µl Washing Solution HSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
a3& jG=!&-I=&8G !&\ /-=(&1!H&1HH&650 µl Washing Solution LSQ&0/2)=&
-I=& 01G& 1!H& 0=!-( E"K=& 1-& D<3<<<&J&K& S"DC3<<<&(GLW& E2(& D&L !3&k )01(H& -I=& $=0= O=(& R".=& N -I& -I=& E /-(1-=3& #/10=& -I=& 8G !& \ /-=(& !-2&1&!=N&C3<&L/&$=0= O=(&R".=3&
& #(2-202/&U4&A)2/1- 2!&2E&O (1/&$*+&E(2L&0=//&0"/-"(=)&SL1J3&U&J&D<^&0=//)W&
&
!!"#$%#&' (")&$*+&, -& A))"=&<?BC<<>& D;&
>3& @=!-( E"K=& 1-& L1J3& )G==H& E2(& C& L !& -2& (=L2O=& 1//& -(10=)& 2E&=-I1!2/3&k )01(H&-I=&C3<&L/&$=0= O=(&R".=3&
D<3#/10=& -I=& 8G !& \ /-=(& !-2& 1& D3U& L/& %/"- 2!& R".=3& @1(=E"//F& 2G=!&-I=&01G&2E&-I=&8G !&\ /-=(&1!H&1HH&60–100 µl RNase-free water3&A!0".1-=& 1-& (22L& -=LG=(1-"(=& E2(& C&L !3& @=!-( E"K=& 1-& ^3<<<&J&K&
S"a3<<<& (GLW& E2(&D&L !3&RN2&=/"- 2!&)-=G)& N -I&=Z"1/&O2/"L=)&2E&$*1)=9E(==& N1-=(& S=3K3&?<&l/&]&?<&l/W& L KI-& !0(=1)=& -I=& F =/H& 2E&=J-(10-=H&O (1/&$*+3&
&
Note
RI=&$*+&01!&.=&=/"-=H& N -I&1& /2N=(&2(&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(& SH=G=!H)& 2!& -I=& =JG=0-=H& F =/H& 2E& $*+W3& %/"- 2!& N -I& /2N=(& O2/9"L=)& 2E& $*1)=9E(==& N1-=(& !0(=1)=)& -I=& E !1/& 02!0=!-(1- 2!& 2E& $*+3&8-2(=&-I=&=J-(10-=H&O (1/&$*+&1-&9C<&V@3&\2(&/2!K&- L=&)-2(1K=&G/10 !K&1-&&9a<&V@& )&(=02LL=!H=H3&
&&
R(2"./=)I22- !K&
&
&
Da& A))"=&<?BC<<>& !!"#$%#&' (")&$*+&, -&
17 Troubleshooting
&
Problem / probable cause Comments and suggestions
Clogged Spin Filter &
! A!)"EE 0 =!-&/F) )&1!HB2(&-22&L"0I&)-1(- !K&L1-=( 1/&
A!0(=1)=&/F) )&- L=3&
A!0(=1)=&0=!-( E"K1- 2!&)G==H3&
+E-=(&/F) )&0=!-( E"K=&-I=&/F)1-=&-2&G=//=-&"!/F)=H&L1-=( 1/3&&
$=H"0=&1L2"!-&2E&)-1(- !K&L1-=( 1/3&
Low amount of extracted viral RNA&
&
! A!)"EE 0 =!-&/F) )& A!0(=1)=&/F) )&- L=3&
$=H"0=&1L2"!-&2E&)-1(- !K&L1-=( 1/3&&
jO=(/21H !K&2E&8G !&\ /-=(&(=H"0=)&F =/HP&
! A!02LG/=-=&=/"- 2!&
&
#(2/2!K&-I=& !0".1- 2!&- L=&N -I&$*1)=9E(==&N1-=(&-2&U&L !"-=)&2(&(=G=1-&=/"- 2!&)-=G&2!0=&1K1 !3&
R1M=&1&I KI=(&O2/"L=&2E&$*1)=9E(==&N1-=(3&
! A!)"EE 0 =!-&L J !K&N -I&6 !H !K&82/"- 2!&$68&
m J&)1LG/=&N -I&6 !H !K&82/"- 2!&$68&.F&G G=-- !K&2(&.F&O2(-=J !K&G( 2(&-2&-(1!)E=(&2E&-I=&)1LG/=&2!-2&-I=&8G !&\ /-=(3&
Low concentration of extracted viral RNA&
&
! R22&L"0I&$*1)=9E(==&N1-=(& %/"-=&-I=&O (1/&$*+&N -I&/2N=(&O2/"L=&2E&$*1)=9E(==&N1-=(3&
! *2&@1(( =(&$*+&1HH=H& +HH&@1(( =(&$*+&-2&-I=&)1LG/=Q&1)&H=9)0( .=H& !&-I=&L1!"1/&1.2O=3&
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