TITLE: RNA extraction and quantitative PCR to assay inflammatory gene expression (Provisional unformatted)
AUTHORS: OLIVIA   HARDING, holzbaur  

[DESCRIPTION]
Real-time quantitative PCR (RT-qPCR) is a sensitive assay to determine the production of selected mRNA transcripts in various conditions. We required such an assay to demonstrate the effects of mitochondrial depolarization in the presence of Parkin, since we found that damaged mitochondria recruited the NF-kB effector complex molecules, NEMO and IKKb. We developed this protocol to test levels of NF-kB response genes in a cell model transiently over-expressing Parkin. With this technique we found significant upregulation of key pro-inflammatory genes normalized to a housekeeping gene, Gapdh. 

[STEPS]
1. -       When working with RNA, take caution to keep space clean to avoid sample degradation by RNases. Clear bench space and wipe with RNaseZap. Change gloves often and wear a mask.
2. -       Use new, sterile supplies of pipet tips and tubes
3. -       Since RNA is vulnerable to degradation, proceed through the extraction and reverse synthase procedures on the same day to avoid storing RNA samples
4. -       Day 1, extract RNA and produce cDNA for all samples for all biological replicates. Day 2, carry out PCR reactions for all replicates.  Materials
5. -      
6. 5 mL capped tubes (Millipore Sigma, EP022364120)
7. -      
8. 2 mL 96-well PCR plates (Amplifit, 1149K06)
9. -       RNaseZap (Sigma, R2020-250ML)  Reagents
10. -       TRIzol (Ambion, 15596018)
11. -       Chloroform
12. -       Isopropanol
13. -       Ethanol
14. -       RNase/DNase free water (Corning, 46-000-CI)
15. -      
16. mM dNTP mix (Invitrogen, 100004893)
17. -       oligo (dT)20 (Life Tech Corp., 58063)
18. -       First-Strand Buffer (Invitrogen, Y02321)
19. -      
20. 1 M DTT (Invitrogen, Y00147)
21. -       RNaseOUT (Invitrogen, 100000840)
22. -       SuperScript III (Invitrogen, 56575)
23. -      
24. 5 M EDTA
25. -      
26. M NaOH
27. -       Oligo Clean and Concentrator Kit (Zymo Research, D4060)
28. -       Primers of interest (see Materials and Methods for the corresponding manuscript for our primer sequences)
29. -       PowerUp SYBRGreen Master Mix (AppliedBiosystems, A25742)  Equipment
30. -       Two user-controlled heat sources (water baths or blocks)
31. -       Equipment to measure concentration of nucleic acids, such as NanoDrop (ThermoFisher 840274200)
32. -       QuantStudio 3 Real-Time PCR System Machine (Applied Biosystems, A28567)  Before Start
33. -       Set one heat source to 60 degrees
34. -       Set one heat source to 50 degrees
35. -       Prepare 75% ethanol with RNase/DNase free water
36. -       The start point for this protocol is after cells grown on 6 cm dishes have been transfected with relevant constructs for 18-24 hours and treated with appropriate small molecules or vehicles. 18-24 hr before collection, transfect 1.5 ug Parkin and .2 ug EGFP-NEMO to 70-80% confluent cells on each 6 cm dish. These should yield ~1 million cells per dish
37. -       For each replicate, one dish was treated with AntA/OligA, one dish was treated with TNFa (positive control), and one dish was treated with vehicle (control) for 5 hr.  Step-by-step  Initial RNA extraction
38. ·      Aspirate media from each dish
39. ·      Add 300 uL cold TRIzol per million cells directly onto the cells and pipet up and down to homogenize
40. ·      transfer to 1.5 mL tube
41. ·      Incubate 5 min, r.t.
42. ·      Add 200 uL chloroform per mL TRIzol
43. ·      Mix by inversion until cloudy homogenous solution
44. ·      Incubate 2-3 min at r.t.
45. ·      Centrifuge 15 min at 12 G, 4 degrees.
46. o   Should separate into red phenol-chloroform (bottom), an organic phase, and colorless aqueous (top)
47. ·      Transfer aqueous phase (top) containing RNA to new tube by angling at 45 degrees and carefully pipetting out. The other phases can be saved for protein or DNA isolation
48. ·      Add 500 uL isopropanol to aqueous phase per 1 mL TRIzol used
49. ·      Incubate 10 min, r.t.
50. ·      Centrifuge 10 min, 12 G at 4 degrees
51. o   RNA will pellet as white, gel-like
52. ·      Discard sup
53. ·      Resuspend pellet in 1 mL 75% EtOH per 1 mL Trizol used
54. ·      Vortex quickly then centrifuge 5 min 7.5 G at 4 degrees
55. ·      Discard sup
56. ·      Air dry pellet 5-10 min
57. o   do not totally dry it; it should start to clarify over drying
58. ·      resuspend the pellet in 50 uL RNase free water by pipetting up and down
59. o   it’s normal if this doesn’t go into suspension
60. ·      incubate at 60 C 10-15 min
61. o   Afterward, set heat bath or block to 65 C
62. ·      measure concentration of RNA with NanoDrop or other     Reverse Transcriptase Reaction to generate cDNA
63. ·      Thaw 5X first-strand buffer and 0.1 M DTT at RT immediately before use. refreeze immediately after.
64. ·      Calculate the volume of each sample needed for 5 ug
65. ·      To 5 ug RNA, add 1 uL 10 mM dNTP Mix (equal parts each base), 1 μl of oligo(dT)20 (50 μM); and sterile water to 13 uL
66. ·      Heat at 65 degrees, 5 min
67. o   Afterward, set heat bath or block to 70 C
68. ·      Incubate on ice 1 min
69. ·      Briefly centrifuge
70. ·      Add 4 uL First-strand buffer, 1 uL 0.1 M DTT, 1 uL RNase OUT inhibitor, 1 uL SuperScript III
71. ·      Gently pipet up and down to mix
72. ·      Incubate at 50 degrees for 45 min
73. o   Afterward, set heat source to 65 C
74. ·      inactivate by heating to 70 degrees for 15 min
75. ·      The result is cDNA.     Clean cDNA (EDTA/NaOH and Zymo Oligo Clean & Conc. Kit)
76. ·      Add 5 uL 0.5 M EDTA and 5 uL 1 M NaOH to each, mix by inversion
77. ·      Heat at 65 C 15 min
78. ·      Adjust volumes to 50 uL with water
79. ·      Add 100 uL Oligo Binding Buffer to each 50 uL
80. ·      Add 400 uL ethanol and mix briefly by pipetting. Transfer to Zymo-Spin Column in the kit
81. ·      Centrifuge 10 G, 30 sec, R.T. and discard the flow through
82. ·      Add 750 uL DNA Wash Buffer to the column
83. ·      Centrifuge 10 G, 30 sec, R.T. and discard the flow through
84. ·      Centrifuge max speed, 1 min, R.T.
85. ·      Transfer the column to a new clean tube and add
86. uL water to the matrix
87. ·      Centrifuge at 10 G, 30 sec, R.T. to elute
88. ·      Measure 260/280 for final conc. The product can be saved at -20.     Set up PCR Reactions     Sample   SYBR Master Mix   Fwd and Rev Primers (10 uM stock to 300 nM final)   cDNA (1:100 dilutions)   Nuclease free water (to 44 uL)     For one reaction (total 11 uL)
89. 5 uL
90. 33 uL
91. ng (this is the maximum mass)   varying
92. -       We use the following worksheet to plan volumes needed for each reaction. The following is our example.     Number of different primer sets = _____8____(p)     Number of replicates per primer set = ___3______(n).      ____8____(p) * ___3______(n) = ____24_____(T) = number of reactions per cDNA sample.     ___24_____(T) * 11 uL = ____264______(V) = volume for each set of cDNA             Replicate   Sample   SYBR Master Mix (V / 2)   cDNA (11 * T ug)   Nuclease free water V – (0.33*n) – (V/2) – cDNA volume   Fwd and Rev Primers (10 uM stock to 300 nM final) (0.33 uL * n) add later     N1   No template control   132   -   130   1 of each     veh     “
93. 2
94. 8   “     TNF     “
95. 5
96. 5   “     AO     “
97. 5
98. 5   “     N2   No template control   “   -   130   “     veh     “
99. 08
100. 9   “     TNF     “
101. 1
102. 9   “     AO     “
103. 07
104. 9   “     N3   No template control   “   -   130   “     veh     “
105. 32
106. 7   “     TNF     “
107. 88
108. 1   “     AO     “
109. 18
110. 8   “
111. -       Mix these then centrifuge quickly
112. -       Split into __8____(p) tubes > (____3____(n) * 10 uL = ____30_____(Pinitial)) in each tube
113. -       add 0.33 uL * n = ___1_____ uL each primer (10 uM) respectively to get total
114. ____32_____(~Pfinal uL)/tube
115. -       Mix again, centrifuge, and add 10 uL each reaction to wells
116. -       Seal the plate with an adhesive cover then centrifuge to get rid of air bubbles and ensure components are combined.
117. -       Can store this at R.T. 24 hr.
118. -       Run the reaction in the QuantStudio with the following procedure     Step   Temp (C)   Duration   Cycles     Cycling Mode                 UDG activation   50
119. min   -     Dual Lock DNA polymerase   95
120. min   -     Denature   95
121. sec   40     Anneal   56*
122. sec     Extend   72
123. min     Dissociation curve                 1
124. 6C/sec to 95
125. sec   -     2
126. 6C/sec to 60
127. min   -     3
128. 15C/sec to 95
129. sec   -     * is variable annealing temp, chosen taking into account the melt curve of all primers
130. -       export all data as an .xls file.
131. -       Analyze with ΔΔ method